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The allocation of resources during bacterial growth is strongly related to the availability of ribosomes and RNA polymerase molecules. Here, coarse-grained models offer a promising start due to their simple structure and the limited number of kinetic parameters. Based on published data sets for proteome and mRNA data in Escherichia coli, and together with mass balance equations describing gene expression, we are able to calculate the number of active molecules (that is, the number of ribosomes that are currently translating nascent and mature mRNA, as well as the number of RNA polymerase molecules on the DNA). This information is a prerequisite for meaningful coarse-grained models. In our approach, the cellular compartment is structured into a cytosolic region and a nucleoid region, and the processes of transcription and translation are assigned accordingly. The theoretical study reveals a quadratic relationship between the number of active ribosomes and the growth rate μ. While the overall available number of ribosomes follows the linear "bacterial growth law", the approach allows us to determine the growth limit for the chosen experimental environment (minimal medium, only one C source). The new approach is in good agreement with published experimental data, and, with a simple rule of thumb can be applied to other cellular systems.

During spliceosome assembly, interactions that bring the 5′ and 3′ ends of an intron in proximity are critical for the production of mature mRNA. Here, we report synergistic roles for the stem-loops 3 (SL3) and 4 (SL4) of the human U1 small nuclear RNA (snRNA) in maintaining the optimal U1 snRNP function, and formation of cross-intron contact with the U2 snRNP. We find that SL3 and SL4 bind distinct spliceosomal proteins and combining a U1 snRNA activity assay with siRNA-mediated knockdown, we demonstrate that SL3 and SL4 act through the RNA helicase UAP56 and the U2 protein SF3A1, respectively. In vitro analysis using UV crosslinking and splicing assays indicated that SL3 likely promotes the SL4-SF3A1 interaction leading to enhancement of A complex formation and pre-mRNA splicing. Overall, these results highlight the vital role of the distinct contacts of SL3 and SL4 in bridging the pre-mRNA bound U1 and U2 snRNPs during the early steps of human spliceosome assembly.

The identification and characterization of rhythmically expressed mRNAs have been an active area of research over the past 20 years, as these mRNAs are believed to produce the daily rhythms in a wide range of biological processes. Circadian transcriptome studies have used mature mRNA as a primary readout and focused largely on rhythmic RNA synthesis as a regulatory mechanism underlying rhythmic mRNA expression. However, RNA synthesis, RNA degradation, or a combination of both must be rhythmic to drive rhythmic RNA profiles, and it is still unclear to what extent rhythmic synthesis leads to rhythmic RNA profiles. In addition, circadian RNA expression is also often tissue specific. Although a handful of genes cycle in all or most tissues, others are rhythmic only in certain tissues, even though the same core clock mechanism is believed to control the rhythmic RNA profiles in all tissues. This review focuses on the dynamics of rhythmic RNA synthesis and degradation and discusses how these steps collectively determine the rhythmicity, phase, and amplitude of RNA accumulation. In particular, we highlight a possible role of RNA degradation in driving tissue-specific RNA rhythms. By unifying findings from experimental and theoretical studies, we will provide a comprehensive overview of how rhythmic gene expression can be achieved and how each regulatory step contributes to tissue-specific circadian transcriptome output in mammals.

The accurate exclusion of introns by RNA splicing is critical for the production of mature mRNA. U2AF1 binds specifically to the 3´ splice site, which includes an essential AG dinucleotide. Even a single amino acid mutation of U2AF1 can cause serious disease such as certain cancers or myelodysplastic syndromes. Here, we describe the first crystal structures of wild-type and pathogenic mutant U2AF1 complexed with target RNA, revealing the mechanism of 3´ splice site selection, and how aberrant splicing results from clinically important mutations. Unexpected features of this mechanism may assist the future development of new treatments against diseases caused by splicing errors., U2AF1 binds to the 3’ splice site of introns and its mutation lead to abnormal splicing. Here the authors solve the crystal structures of wild type and pathogenic mutant U2AF1 bound to target RNA, showing that different target sequence is preferred by pathogenic mutant.

In eukaryotes, mature mRNA is formed through modifications of precursor mRNA, one of which is 5' cap biosynthesis, involving RNA cap guanine-N7 methyltransferase (N7-MTase). N7-MTases are also encoded by some eukaryotic viruses and facilitate their replication. N7-MTase inhibitors have therapeutic potential, but their discovery is difficult because long RNA substrates are usually required for activity. Herein, we report a universal N7-MTase activity assay based on small-molecule fluorescent probes. We synthesized 12 fluorescent substrate analogues (GpppA and GpppG derivatives) varying in the dye type, dye attachment site, and linker length. GpppA labeled with pyrene at the 3'-O position of adenosine acted as an artificial substrate with the properties of a turn-off probe for all three tested N7-MTases (human, parasite, and viral). Using this compound, a N7-MTase inhibitor assay adaptable to high-throughput screening was developed and used to screen synthetic substrate analogues and a commercial library. Several inhibitors with nanomolar activities were identified.

The spliceosome removes introns from messenger RNA precursors (pre-mRNA). Decades of biochemistry and genetics combined with recent structural studies of the spliceosome have produced a detailed view of the mechanism of splicing. In this review, we aim to make this mechanism understandable and provide several videos of the spliceosome in action to illustrate the intricate choreography of splicing. The U1 and U2 small nuclear ribonucleoproteins (snRNPs) mark an intron and recruit the U4/U6.U5 tri-snRNP. Transfer of the 5′ splice site (5′SS) from U1 to U6 snRNA triggers unwinding of U6 snRNA from U4 snRNA. U6 folds with U2 snRNA into an RNA-based active site that positions the 5′SS at two catalytic metal ions. The branch point (BP) adenosine attacks the 5′SS, producing a free 5′ exon. Removal of the BP adenosine from the active site allows the 3′SS to bind, so that the 5′ exon attacks the 3′SS to produce mature mRNA and an excised lariat intron.

Significance The random nature of gene expression is well established experimentally. Mathematical modeling provides a means of understanding the factors leading to the observed stochasticity. In this article, we extend the classical two-state model of stochastic mRNA dynamics to include a considerable number of salient features of single-cell biology, such as cell division, replication, mRNA maturation, dosage compensation, and growth-dependent transcription. By means of biologically relevant approximations, we obtain expressions for the time-dependent distributions of mRNA and protein numbers. These provide insight into how fluctuations are modified and controlled by complex intracellular processes., The stochasticity of gene expression presents significant challenges to the modeling of genetic networks. A two-state model describing promoter switching, transcription, and messenger RNA (mRNA) decay is the standard model of stochastic mRNA dynamics in eukaryotic cells. Here, we extend this model to include mRNA maturation, cell division, gene replication, dosage compensation, and growth-dependent transcription. We derive expressions for the time-dependent distributions of nascent mRNA and mature mRNA numbers, provided two assumptions hold: 1) nascent mRNA dynamics are much faster than those of mature mRNA; and 2) gene-inactivation events occur far more frequently than gene-activation events. We confirm that thousands of eukaryotic genes satisfy these assumptions by using data from yeast, mouse, and human cells. We use the expressions to perform a sensitivity analysis of the coefficient of variation of mRNA fluctuations averaged over the cell cycle, for a large number of genes in mouse embryonic stem cells, identifying degradation and gene-activation rates as the most sensitive parameters. Furthermore, it is shown that, despite the model’s complexity, the time-dependent distributions predicted by our model are generally well approximated by the negative binomial distribution. Finally, we extend our model to include translation, protein decay, and auto-regulatory feedback, and derive expressions for the approximate time-dependent protein-number distributions, assuming slow protein decay. Our expressions enable us to study how complex biological processes contribute to the fluctuations of gene products in eukaryotic cells, as well as allowing a detailed quantitative comparison with experimental data via maximum-likelihood methods.

Transcriptional rates are often estimated by fitting the distribution of mature mRNA numbers measured using smFISH (single molecule fluorescence in situ hybridization) with the distribution predicted by the telegraph model of gene expression, which defines two promoter states of activity and inactivity. However, fluctuations in mature mRNA numbers are strongly affected by processes downstream of transcription. In addition, the telegraph model assumes one gene copy, but in experiments cells may have two gene copies as cells replicate their genome during the cell cycle. Whilst it is often presumed that post-transcriptional noise and gene copy number variation affect transcriptional parameter estimation, the size of the error introduced remains unclear. To address this issue, here we measure both mature and nascent mRNA distributions of GAL10 in yeast cells using smFISH and classify each cell according to its cell cycle phase. We infer transcriptional parameters from mature and nascent mRNA distributions, with and without accounting for cell cycle phase and compare the results to live-cell transcription measurements of the same gene. We find that: (i) correcting for cell cycle dynamics decreases the promoter switching rates and the initiation rate, and increases the fraction of time spent in the active state, as well as the burst size; (ii) additional correction for post-transcriptional noise leads to further increases in the burst size and to a large reduction in the errors in parameter estimation. Furthermore, we outline how to correctly adjust for measurement noise in smFISH due to uncertainty in transcription site localisation when introns cannot be labelled. Simulations with parameters estimated from nascent smFISH data, which is corrected for cell cycle phases and measurement noise, leads to autocorrelation functions that agree with those obtained from live-cell imaging.

Simple Summary Splicing is an important mechanism by which precursor mRNA is modified into mature mRNA. This splicing plays a major role in the generation of different proteins required for cells. Cancer cells modulate this splicing in such a way that it facilitates uncontrolled growth and survival. Cancer is one of the leading causes of death, and the therapies that are currently available also affect normal cells. Antisense oligonucleotides (AOs) are synthetic DNA/RNA that bind specifically to target mRNA and thereby have fewer off-target effects. These AOs have the potential to modulate the splicing mechanism. In this review, we will discuss the different modes of action of AOs and their potential in targeting cancer. Abstract Splicing is an essential process wherein precursor messenger RNA (pre-mRNA) is reshaped into mature mRNA. In alternative splicing, exons of any pre-mRNA get rearranged to form mRNA variants and subsequently protein isoforms, which are distinct both by structure and function. On the other hand, aberrant splicing is the cause of many disorders, including cancer. In the past few decades, developments in the understanding of the underlying biological basis for cancer progression and therapeutic resistance have identified many oncogenes as well as carcinogenic splice variants of essential genes. These transcripts are involved in various cellular processes, such as apoptosis, cell signaling and proliferation. Strategies to inhibit these carcinogenic isoforms at the mRNA level are promising. Antisense oligonucleotides (AOs) have been developed to inhibit the production of alternatively spliced carcinogenic isoforms through splice modulation or mRNA degradation. AOs can also be used to induce splice switching, where the expression of an oncogenic protein can be inhibited by the induction of a premature stop codon. In general, AOs are modified chemically to increase their stability and binding affinity. One of the major concerns with AOs is efficient delivery. Strategies for the delivery of AOs are constantly being evolved to facilitate the entry of AOs into cells. In this review, the different chemical modifications employed and delivery strategies applied are discussed. In addition to that various AOs in clinical trials and their efficacy are discussed herein with a focus on six distinct studies that use AO-mediated exon skipping as a therapeutic strategy to combat cancer.

Funding Information: S.B. and R.G. were supported by a Leverhulme Trust grant no. (RPG-2018-423). J.H. was supported by a BBSRC EASTBIO PhD studentship. Two-state models (telegraph-like models) have a successful history of predicting distributions of cellular and nascent mRNA numbers that can well fit experimental data. These models exclude key rate limiting steps, and hence it is unclear why they are able to accurately predict the number distributions. To answer this question, here we compare these models to a novel stochastic mechanistic model of transcription in mammalian cells that presents a unified description of transcriptional factor, polymerase and mature mRNA dynamics. We show that there is a large region of parameter space where the first, second and third moments of the distributions of the waiting times between two consecutively produced transcripts (nascent or mature) of two-state and mechanistic models exactly match. In this region: (i) one can uniquely express the two-state model parameters in terms of those of the mechanistic model, (ii) the models are practically indistinguishable by comparison of their transcript numbers distributions, and (iii) they are distinguishable from the shape of their waiting time distributions. Our results clarify the relationship between different gene expression models and identify a means to select between them from experimental data. Publisher PDF

RNA splicing, the process by which precursor mRNA (pre-mRNA) is processed to mature mRNA, is catalyzed by the spliceosome. Recently, Chatrikhi et al. identified pharmacologic means to perturb splicing by enhancing the spliceosome's binding to pre-mRNA. This represents a novel chemical target and mechanism for therapeutic modulation of splicing.

Alternative pre-mRNA splicing has long been proposed to contribute greatly to proteome complexity. However, the extent to which mature mRNA isoforms are successfully translated into protein remains controversial. Here, we used high-throughput RNA sequencing and mass spectrometry (MS)–based proteomics to better evaluate the translation of alternatively spliced mRNAs. To increase proteome coverage and improve protein quantitation, we optimized cell fractionation and sample processing steps at both the protein and peptide level. Furthermore, we generated a custom peptide database trained on analysis of RNA-seq data with MAJIQ, an algorithm optimized to detect and quantify differential and unannotated splice junction usage. We matched tandem mass spectra acquired by data-dependent acquisition (DDA) against our custom RNA-seq based database, as well as SWISS-PROT and RefSeq databases to improve identification of splicing-derived proteoforms by 28% compared with use of the SWISS-PROT database alone. Altogether, we identified peptide evidence for 554 alternate proteoforms corresponding to 274 genes. Our increased depth and detection of proteins also allowed us to track changes in the transcriptome and proteome induced by T-cell stimulation, as well as fluctuations in protein subcellular localization. In sum, our data here confirm that use of generic databases in proteomic studies underestimates the number of spliced mRNA isoforms that are translated into protein and provides a workflow that improves isoform detection in large-scale proteomic experiments.

Most mitochondrial mRNAs are transcribed as polycistronic precursors that are cleaved by endonucleases to produce mature mRNA transcripts. However, recent studies have shown that mitochondrial transcripts in the kinetoplastid protozoan, Trypanosoma brucei, are transcribed individually. Also unlike most mitochondrial mRNAs, the 5′ end of these transcripts harbor a triphosphate that is hydrolyzed. This modification is carried out by a putative Nudix hydrolase called MERS1. The Nudix motif in MERS1 is degenerate, lacking a conserved glutamic acid, thus it is unclear how it may bind its substrates and whether it contains a Nudix fold. To obtain insight into this unusual hydrolase, we determined structures of apo, GTP-bound and RNA-bound T. brucei MERS1 to 2.30 Å, 2.45 Å, and 2.60 Å, respectively. The MERS1 structure has a unique fold that indeed contains a Nudix motif. The nucleotide bound structures combined with binding studies reveal that MERS1 shows preference for RNA sequences with a central guanine repeat which it binds in a single-stranded conformation. The apo MERS1 structure indicates that a significant portion of its nucleotide binding site folds upon substrate binding. Finally, a potential interaction region for a binding partner, MERS2, that activates MERS1 was identified. The MERS2-like peptide inserts a glutamate near the missing Nudix acidic residue in the RNA binding pocket, suggesting how the enzyme may be activated. Thus, the combined studies reveal insight into the structure and enzyme properties of MERS1 and its substrate-binding activities.

PARP1 is an abundant nuclear protein with many pleiotropic functions involved in epigenetic and transcriptional controls. Abundance of mRNA depends on the balance between synthesis and decay of a particular transcript. PARP1 binds RNA and its depletion results in increased expression of genes involved in nonsense-mediated decay, suggesting that PARP1 might be involved in mRNA stability. This is of interest considering RNA binding proteins play key roles in post-transcriptional processes in all eukaryotes. We tested the direct impact of PARP1 and PARylation on mRNA stability and decay. By measuring the half-lives of two PARP1-mRNA targets we found that the half-lives were significantly decreased in PARP1-depleted cells. PARP1 depletion impacted both the synthesis of nascent mRNA and the stability of mature mRNAs. PARylation impacted the production of nascent mRNA and the stability of mature mRNA, albeit to a lesser extent than PARP1 KD. PARylation enhanced the impact of PARP1 depletion. These studies provide the first direct comparative role of PARP1 and PARylation in RNA stability and decay, adding a new dimension as to how PARP1 regulates gene expression. These studies present a platform to begin to tease out the influence of PARP1 at each step of RNA biogenesis and decay to fine-tune gene expression.

The crustacean hyperglycemic hormone (CHH) gene of Portunus trituberculatus (Pt-CHH) consists of four exons and three introns spanning 3849 bp in size and generating two mature mRNA, Pt-CHH1, and Pt-CHH2. The primary gene transcript produces a cDNA encoding for the putative Pt-CHH2 from exons 1, 2, 3, and 4 and an alternative transcript encodes for a putative Pt-CHH1 peptide from exons 1, 2, and 4. A promoter fragment of about 3 kb was obtained by genomic walking. The tissue-specific expression pattern is examined by reverse transcriptase chain reaction, and the results show that Pt-CHH1 is detected in the eyestalk, brain, muscle, and blood. However, Pt-CHH2 is detected in the ganglia thoracalis and gill. The results indicate that the expression of Pt-CHH2 in the gill might suggest a potential role in osmoregulation. The Pt-CHH transcript level in the gill increases when the crab is exposed to low salinity. The injection of dsRNA for Pt-CHH causes a significant reduction in Pt-CHH2 transcript level and the activity of Na(+)/K(+)-ATPase, and carbonic anhydrase (CA) show a serious decrease. In conclusion, this study provides molecular evidence to support the osmoregulatory function of Pt-CHH2.

The poly(A) RNA binding Zn finger ribonucleoprotein Nab2 functions to control the length of 3' poly(A) tails in Saccharomyces cerevisiae as well as contributing to the integration of the nuclear export of mature mRNA with preceding steps in the nuclear phase of the gene expression pathway. Nab2 is constructed from an N-terminal PWI-fold domain, followed by QQQP and RGG motifs and then seven CCCH Zn fingers. The nuclear pore-associated proteins Gfd1 and Mlp1 bind to opposite sides of the Nab2 N-terminal domain and function in the nuclear export of mRNA, whereas the Zn fingers, especially fingers 5-7, bind to A-rich regions of mature transcripts and function to regulate poly(A) tail length as well as mRNA compaction prior to nuclear export. Nab2 Zn fingers 5-7 have a defined spatial arrangement, with fingers 5 and 7 arranged on one side of the cluster and finger 6 on the other side. This spatial arrangement facilitates the dimerization of Nab2 when bound to adenine-rich RNAs and regulates both the termination of 3' polyadenylation and transcript compaction. Nab2 also functions to coordinate steps in the nuclear phase of the gene expression pathway, such as splicing and polyadenylation, with the generation of mature mRNA and its nuclear export. Nab2 orthologues in higher Eukaryotes have similar domain structures and play roles associated with the regulation of splicing and polyadenylation. Importantly, mutations in the gene encoding the human Nab2 orthologue ZC3H14 and cause intellectual disability.

After synthesis and processing in the nucleus, mature mRNA penetrates through nuclear pores to cytoplasm. mRNP particles are formed in the nucleus and remodeled in cytoplasm, resulting in association with factors required for mRNP proper functions. In the cytoplasmic compartment, mRNP particles can include proteins participating in nuclear interactions, as well as new factors specific for cytoplasmic mRNA–protein interactions. Functions of newly synthesized mRNAs and their cytoplasmic localization are substantially diverse. Cytoskeletal and motor proteins were shown to participate in delivery of mRNP particles to their localization sites. Several families of motor proteins are known in eukaryotes, and functions of all of them were shown to be coordinated in order to compose machinery for cargo delivery to specific cytoplasmic destinations. Adapter proteins, mediators between mRNP particles and motor proteins, are also required to make this transport system functional. Unlike the well-studied system of the cytoskeletal and motor proteins, our knowledge of how mRNP particles bind motor proteins via adapter ones is still sparse.

Cytidine-to-uridine (C-to-U) RNA editing has been generally observed in land plants; however, reverse (U-to-C) RNA editing is a rare phenomenon. In this study, we investigated the U-to-C RNA editing-related genes in Arabidopsis tissues and the effects on mRNA stability, with a special focus on PPR proteins. A previous study showed the extensive occurrence of U-to-C RNA editing in 12-day and 20-dayold Arabidopsis seedlings. Here, we have demonstrated the effects of this "reverse" RNA editing on the mRNA stability for all seven edited genes. We also identified U-to-C RNA editing in the nuclear PPR gene (AT2G19280) in 12-day-old seedlings of Arabidopsis thaliana. The U-to-C RNA editing sites were found in the untranslated region (3' UTR) of the mature mRNA and may affect its secondary structure. We also examined the correlation between U-to-C RNA editing-related genes and their mRNA abundance. Furthermore, we investigated the effects of U-to-C RNA editing in Arabidopsis using the transcription inhibitor actinomycin D (Act D). The addition of Act D to the seedlings of transgenic Arabidopsis generated by Agrobacterium-mediated transformation showed that single nucleotide base conversion adversely affected the mRNA secondary structure and stability.

How DNA sequence affects the dynamics and position of RNA Polymerase II (Pol II) during transcription remains poorly understood. Here we used naturally occurring genetic variation in F1 hybrid mice to explore how DNA sequence differences affect the genome-wide distribution of Pol II. We measured the position and orientation of Pol II in eight organs collected from heterozygous F1 hybrid mice using ChRO-seq. Our data revealed a strong genetic basis for the precise coordinates of transcription initiation and promoter proximal pause, allowing us to redefine molecular models of core transcriptional processes. Our results implicate the strength of base pairing between A-T or G-C dinucleotides as key determinants to the position of Pol II initiation and pause. We report evidence that initiation site selection follows a stochastic process similar to brownian motion along the DNA template. We found widespread differences in the position of transcription termination, which impact the primary structure and stability of mature mRNA. Finally, we report evidence that allelic changes in transcription often affect mRNA and ncRNA expression across broad genomic domains. Collectively, we reveal how DNA sequences shape core transcriptional processes at single nucleotide resolution in mammals.

Precursor messenger RNA (pre-mRNA) splicing is a fundamental step in eukaryotic gene expression that systematically removes non-coding regions (introns) and ligates coding regions (exons) into a continuous message (mature mRNA). This process is highly regulated and can be highly flexible through a process known as alternative splicing, which allows for several transcripts to arise from a single gene, thereby greatly increasing genetic plasticity and the diversity of proteome. Alternative splicing is particularly prevalent in neuronal cells, where the splicing patterns are continuously changing to maintain cellular homeostasis and promote neurogenesis, migration and synaptic function. The continuous changes in splicing patterns and a high demand on many cis- and trans-splicing factors contribute to the susceptibility of neuronal tissues to splicing defects. The resultant neurodegenerative diseases are a large group of disorders defined by a gradual loss of neurons and a progressive impairment in neuronal function. Several of the most common neurodegenerative diseases involve some form of splicing defect(s), such as Alzheimer’s disease, Parkinson’s disease and spinal muscular atrophy. Our growing understanding of RNA splicing has led to the explosion of research in the field of splice-switching antisense oligonucleotide therapeutics. Here we review our current understanding of the effects alternative splicing has on neuronal differentiation, neuronal migration, synaptic maturation and regulation, as well as the impact on neurodegenerative diseases. We will also review the current landscape of splice-switching antisense oligonucleotides as a therapeutic strategy for a number of common neurodegenerative disorders.

As evidences showed that UOX(Gene ID: 391051), a single pseudogene formed after multiple mutations during human evolution, could be transcribed to mature mRNA and translated to two short peptides, we hypothesized that urate oxidase with higher homology with deduced human urate oxidase (dHU) might have lower immunogenicity. In this work, we constructed a "resurrected" human-source urate oxidase (rHU19) based on dHU. It obtained better uricolytic activity (8.29 U/mg) and catalytic efficiency (3.32 s-1 μM-1) compared with wild porcine urate oxidase (wPU) and FDA-approved porcine-baboon chimera (PBC). Maintaining high homology with dHU (93.75 %), rHU19 could be more suitable for the treatment of gout and hyperuricemia theoretically.

Exon skipping is a disease-modifying therapy in which oligonucleotide analogues mask specific exons, eliminating them from the mature mRNA, and also the cognate protein. That is one possible therapeutic aim, but it can also be used to restore the reading frame for diseases caused by frameshift mutations, which is the case for Duchenne muscular dystrophy (DMD). DMD most commonly arises as a result of large exonic deletions that create a frameshift and abolish protein expression. Loss of dystrophin protein leads to the pathology of the disease, which is severe, causing death generally in the second or third decade of life. Here, the primary aim of exon skipping is restoration of protein expression by reading frame correction. However, the therapeutically expressed protein is missing both the region of the underlying genetic defect and the therapeutically skipped exon. How removing some region from the middle of a protein affects its structure and function is unclear. Many different underlying deletions are known, and exon skipping can be applied in many ways, in some cases in different ways to the same defect. These vary in how severely perturbative they are, with possible clinical consequences. In this study, we examine a systematic, comprehensive panel of exon edits in a region of dystrophin and identify for the first time exon edits that are minimally perturbed and appear to keep the structural stability similar to that of wild-type protein. We also identify factors that appear to be correlated with how perturbative an edit is.

Synthetic messenger RNA (mRNA)-based therapeutics are an increasingly popular approach to gene and cell therapies, genome engineering, enzyme replacement therapy, and now, during the global SARS-CoV-2 pandemic, vaccine development. mRNA for such purposes can be synthesized through an enzymatic in vitro transcription (IVT) reaction and formulated for in vivo delivery. Mature mRNA requires a 5'-cap for gene expression and mRNA stability. There are two methods to add a cap in vitro: via a two-step multi-enzymatic reaction or co-transcriptionally. Co-transcriptional methods minimize reaction steps and enzymes needed to make mRNA when compared to enzymatic capping. CleanCap® AG co-transcriptional capping results in 5 mg/ml of IVT with 94% 5'-cap 1 structure. This is highly efficient compared to first-generation cap analogs, such as mCap and ARCA, that incorporate cap 0 structures at lower efficiency and reaction yield. This article describes co-transcriptional capping using TriLink Biotechnology's CleanCap® AG in IVT. © 2021 Wiley Periodicals LLC. Basic Protocol 1: IVT with CleanCap Basic Protocol 2: mRNA purification and analysis.

Motivation Alternative splicing removes intronic sequences from pre-mRNAs in alternative ways to produce different forms (isoforms) of mature mRNA. The composition of expressed transcripts gives specific functionalities to cells in a particular condition or developmental stage. In addition, a large fraction of human disease mutations affect splicing and lead to aberrant mRNA and protein products. Current methods that interrogate the transcriptome based on RNA-seq either suffer from short-read length when trying to infer full-length transcripts, or are restricted to predefined units of alternative splicing that they quantify from local read evidence. Results Instead of attempting to quantify individual outcomes of the splicing process such as local splicing events or full-length transcripts, we propose to quantify alternative splicing using a simplified probabilistic model of the underlying splicing process. Our model is based on the usage of individual splice sites and can generate arbitrarily complex types of splicing patterns. In our implementation, McSplicer, we estimate the parameters of our model using all read data at once and we demonstrate in our experiments that this yields more accurate estimates compared to competing methods. Our model is able to describe multiple effects of splicing mutations using few, easy to interpret parameters, as we illustrate in an experiment on RNA-seq data from autism spectrum disorder patients. Availability and implementation McSplicer source code is available at https://github.com/canzarlab/McSplicer and has been deposited in archived format at https://doi.org/10.5281/zenodo.4449881. Supplementary information Supplementary data are available at Bioinformatics online.

The structure of eukaryotic genes is generally a combination of exons interrupted by intragenic non-coding DNA regions (introns) removed by RNA splicing to generate the mature mRNA. A fraction of genes, however, comprise a single coding exon with introns in their untranslated regions or are intronless genes (IGs), lacking introns entirely. The latter code for essential proteins involved in development, growth, and cell proliferation and their expression has been proposed to be highly specialized for neuro-specific functions and linked to cancer, neuropathies, and developmental disorders. The abundant presence of introns in eukaryotic genomes is pivotal for the precise control of gene expression. Notwithstanding, IGs exempting splicing events entail a higher transcriptional fidelity, making them even more valuable for regulatory roles. This work aimed to infer the functional role and evolutionary history of IGs centered on the mouse genome. IGs consist of a subgroup of genes with one exon including coding genes, non-coding genes, and pseudogenes, which conform approximately 6% of a total of 21,527 genes. To understand their prevalence, biological relevance, and evolution, we identified and studied 1,116 IG functional proteins validating their differential expression in transcriptomic data of embryonic mouse telencephalon. Our results showed that overall expression levels of IGs are lower than those of MEGs. However, strongly up-regulated IGs include transcription factors (TFs) such as the class 3 of POU (HMG Box), Neurog1, Olig1, and BHLHe22, BHLHe23, among other essential genes including the β-cluster of protocadherins. Most striking was the finding that IG-encoded BHLH TFs fit the criteria to be classified as microproteins. Finally, predicted protein orthologs in other six genomes confirmed high conservation of IGs associated with regulating neural processes and with chromatin organization and epigenetic regulation in Vertebrata. Moreover, this study highlights that IGs are essential modulators of regulatory processes, such as the Wnt signaling pathway and biological processes as pivotal as sensory organ developing at a transcriptional and post-translational level. Overall, our results suggest that IG proteins have specialized, prevalent, and unique biological roles and that functional divergence between IGs and MEGs is likely to be the result of specific evolutionary constraints.

Summary The lack of molecular diagnoses in rare genetic diseases can be explained by limitations of current standard genomic technologies. Upcoming long-read techniques have complementary strengths to overcome these limitations, with a particular strength in identifying structural variants. By using optical genome mapping and long-read sequencing, we aimed to identify the pathogenic variant in a large family with X-linked choroideremia. In this family, aberrant splicing of exon 12 of the choroideremia gene CHM was detected in 2003, but the underlying genomic defect remained elusive. Optical genome mapping and long-read sequencing approaches now revealed an intragenic 1,752 bp inverted duplication including exon 12 and surrounding regions, located downstream of the wild-type copy of exon 12. Both breakpoint junctions were confirmed with Sanger sequencing and segregate with the X-linked inheritance in the family. The breakpoint junctions displayed sequence microhomology suggestive for an erroneous replication mechanism as the origin of the structural variant. The inverted duplication is predicted to result in a hairpin formation of the pre-mRNA with the wild-type exon 12, leading to exon skipping in the mature mRNA. The identified inverted duplication is deemed the hidden pathogenic cause of disease in this family. Our study shows that optical genome mapping and long-read sequencing have significant potential for the identification of (hidden) structural variants in rare genetic diseases., In a family with X-linked choroideremia, aberrant splicing of the gene CHM was detected previously, but the underlying genomic defect remained elusive. Long-read technologies revealed an inverted duplication as the hidden pathogenic cause of disease and thereby show significant potential for the identification of (hidden) SVs in rare genetic diseases.

Marfan syndrome (MFS) is one of the most common hereditary connective tissue diseases, with great individual heterogeneity. We reported a Chinese pregnancy with Clinical diagnosis of MFS, performed whole-exome sequencing, and screened for the genetic abnormality. We also conducted an in vitro mini-gene splicing assay to demonstrate the predicted harmful effects of an intronic variant of FBN-1. Exome sequencing identified a novel intronic variant (c.6497-13 T>A) in intron 53 of the FBN-1 gene (NM_000138.4). It’s predicted to insert 11 bp of intron 53 into the mature mRNA. The mini-gene splicing experiment demonstrated that c.6497-13 T>A could result in 11 bp retention in intron 53 to exon 54 (c.6496_6497ins gtttcttgcag) and the use of an alternative donor causing the frameshift p.Asp2166Glyfs*23. According to the results, the pregnant woman chose to continue the pregnancy and gave birth to a healthy baby. This study expands the genetic mutation spectrum of MFS patients and indicates the importance of intron sequencing. Supplementary Information The online version contains supplementary material available at 10.1186/s41065-020-00170-w.

As one of the widely occurring RNA modifications, 5-methyluridine (m5U) has recently been shown to play critical roles in various biological functions and disease pathogenesis, such as under stress response and during breast cancer development. Precise identification of m5U sites on RNA is vital for the understanding of the regulatory mechanisms of RNA life. We present here m5UPred, the first web server for in silico identification of m5U sites from the primary sequences of RNA. Built upon the support vector machine (SVM) algorithm and the biochemical encoding scheme, m5UPred achieved reasonable prediction performance with the area under the receiver operating characteristic curve (AUC) greater than 0.954 by 5-fold cross-validation and independent testing datasets. To critically test and validate the performance of our newly proposed predictor, the experimentally validated m5U sites were further separated by high-throughput sequencing techniques (miCLIP-Seq and FICC-Seq) and cell types (HEK293 and HAP1). When tested on cross-technique and cross-cell-type validation using independent datasets, m5UPred achieved an average AUC of 0.922 and 0.926 under mature mRNA mode, respectively, showing reasonable accuracy and reliability. The m5UPred web server is freely accessible now and it should make a useful tool for the researchers who are interested in m5U RNA modification., Graphical Abstract, Jie Jiang et al. present m5UPred, the first web server for identification of m5U sites from the primary sequences of RNA by advanced computational method in this study. It can help to accurately predict the m5U sites from RNA sequences, which will assist researchers to understand the RNA methylation mechanism and its biological functions.

The export of mRNA from nucleus to cytoplasm requires the conserved and essential transcription and export (TREX) complex (THO–UAP56/DDX39B–ALYREF). TREX selectively binds mRNA maturation marks and licenses mRNA for nuclear export by loading the export factor NXF1–NXT1. How TREX integrates these marks and achieves high selectivity for mature mRNA is poorly understood. Here, we report the cryo-electron microscopy structure of the human THO–UAP56/DDX39B complex at 3.3 Å resolution. The seven-subunit THO–UAP56/DDX39B complex multimerizes into a 28-subunit tetrameric assembly, suggesting that selective recognition of mature mRNA is facilitated by the simultaneous sensing of multiple, spatially distant mRNA regions and maturation marks. Two UAP56/DDX39B RNA helicases are juxtaposed at each end of the tetramer, which would allow one bivalent ALYREF protein to bridge adjacent helicases and regulate the TREX–mRNA interaction. Our structural and biochemical results suggest a conserved model for TREX complex function that depends on multivalent interactions between proteins and mRNA., eLife digest The DNA of human and other eukaryotic cells is stored inside a compartment called the nucleus. DNA carries the genetic code and provides a blueprint for all of the cell’s proteins. However, protein production occurs outside the nucleus, in the main body of the cell. To transmit genetic information from one compartment to the other, the DNA sequences are first transcribed into another molecule called messenger RNA, or mRNA for short. Once made, mRNA exits the nucleus and enters the cell’s main body to encounter the machinery that translates its sequence into a protein. Before mRNA can exit the nucleus, it must first undergo a series of modifications, which result in the mRNA molecule being successively bound to specific proteins. Once mRNA has passed through these steps, it is recognized by the transcription-and-export complex, or TREX for short, which is comprised of several proteins. When TREX binds to mRNA, it adds on a final protein which allows the mRNA molecule to be transported out of the nucleus. However, it remained unclear how TREX selects the completed mRNA-protein complexes that are ready for export while at the same time recognizing the wide variety of mRNA molecules produced by cells. Now, Pühringer and Hohmann et al. have identified the first three-dimensional structure of the core of the human TREX complex using a technique called cryo-electron microscopy. This revealed that the seven proteins of the TREX core assemble into a large complex that has four copies of each protein. The structure suggests that TREX can bind to mRNA and its attached proteins in various ways. These different binding arrangements may help the complex select which mRNA molecules are fully modified and ready to be exported. The structure also sheds light on how mutations in this complex can lead to diseases such as Beaulieu–Boycott–Innes syndrome (BBIS). This work will help guide future research into the activity of TREX, including how its structure changes when it binds to mRNA and deposits the final transport protein. Identifying these structures will make it easier to design experiments that target specific aspects of TREX activity and provide new insights into how these complexes work.

Intron retention (IR) occurs when a complete and unspliced intron remains in mature mRNA. An increasing body of literature has demonstrated a major role for IR in numerous biological functions, including several that impact human health and disease. Although experimental technologies used to study other forms of mRNA splicing can also be used to investigate IR, a specialized downstream computational analysis is optimal for IR discovery and analysis. Here we provide a review of IR and its biological implications, as well as a practical guide for how to detect and analyze it. Several methods, including long read third generation direct RNA sequencing, are described. We have developed an R package, FakIR, to facilitate the execution of the bioinformatic tasks recommended in this review and a tutorial on how to fit them to users aims. Additionally, we provide guidelines and experimental protocols to validate IR discovery and to evaluate the potential impact of IR on gene expression and protein output. This article is categorized under: RNA Evolution and Genomics > Computational Analyses of RNA RNA Processing > Splicing Regulation/Alternative Splicing RNA Methods > RNA Analyses in vitro and In Silico.

Exon skipping is a disease modifying therapy that operates at the RNA level. In this strategy, oligonucleotide analog drugs are used to specifically mask specific exons and prevent them from inclusion in the mature mRNA. Of course, this also results in loss of the corresponding region from the cognate protein, which is one possible therapeutic aim. Exon skipping can also be used to restore protein expression in cases where a genetic frameshift mutation has occurred, and this how it is applied to Duchenne muscular dystrophy, DMD. DMD most commonly arises as a result of large exonic deletions that juxtapose flanking exons of incompatible reading frame in the dystrophin gene, creating a frameshift and abolishing protein expression. Loss of dystrophin protein leads to the pathology of the disease, which is severe, causing death generally in the second or third decade of life. Here, the primary aim of exon skipping is the restoration of the reading frame by skipping an exon adjacent to the patient’s original defect. However, the therapeutically expressed protein is of course edited, and missing both the region of the underlying genetic defect, as well as the therapeutically skipped exon. While restoring some protein expression is good, how removing some region from the middle of a protein effects its structure and function is unclear. Complicating this in the case of DMD is the fact that the dystrophin gene is very large, containing 79 exons. Many different underlying deletions are known, and exon skipping can be applied in many ways. It has previously been shown that many exon-skip edits result in structural perturbations of varying degrees. What has been unclear is whether and how exon editing can be done to minimize these perturbations. In this study we examine a systematic and comprehensive panel of possible exon edits in a region of the dystrophin protein, and identify for the first time, exon edits that appear to maintain structural stability similar to wildtype protein. We also identify factors that appear to be correlated with the degree of structural perturbation, such as the number of cooperative protein domains, as well as how the underlying exon structure interacts with the protein domain structure.

RNA-seq datasets can contain millions of intron reads per library that are typically removed from downstream analysis. Only reads overlapping annotated exons are considered to be informative since mature mRNA is assumed to be the major component sequenced, especially for poly(A) RNA libraries. In this study, we show that intron reads are informative, and through exploratory data analysis of read coverage that intron signal is representative of both pre-mRNAs and intron retention. We demonstrate how intron reads can be utilized in differential expression analysis using our index method where a unique set of differentially expressed genes can be detected using intron counts. In exploring read coverage, we also developed the superintronic software that quickly and robustly calculates user-defined summary statistics for exonic and intronic regions. Across multiple datasets, superintronic enabled us to identify several genes with distinctly retained introns that had similar coverage levels to that of neighbouring exons. The work and ideas presented in this paper is the first of its kind to consider multiple biological sources for intron reads through exploratory data analysis, minimizing bias in discovery and interpretation of results. Our findings open up possibilities for further methods development for intron reads and RNA-seq data in general.

N6-methyladenosine (m6A) is the most abundant post-transcriptional modification in mRNA, and regulates critical biological functions via m6A reader proteins that bind to m6A-containing transcripts. There exist multiple m6A reader proteins in the human genome, but their respective binding specificity and functional relevance under different biological contexts are not yet fully understood due to the limitation of experimental approaches. An in silico study was devised to unveil the target specificity and regulatory functions of different m6A readers. We established a support vector machine-based computational framework to predict the epitranscriptome-wide targets of six m6A reader proteins (YTHDF1-3, YTHDC1-2, and EIF3A) based on 58 genomic features as well as the conventional sequence-derived features. Our model achieved an average AUC of 0.981 and 0.893 under the full-transcript and mature mRNA model, respectively, marking a substantial improvement in accuracy compared to the sequence encoding schemes tested. Additionally, the distinct biological characteristics of each individual m6A reader were explored via the distribution, conservation, Gene Ontology enrichment, cellular components and molecular functions of their target m6A sites. A web server was constructed for predicting the putative binding readers of m6A sites to serve the research community, and is freely accessible at: http://m6areader.rnamd.com.

Morphogen signaling contributes to the proper spatiotemporal expression of genes during development. One method of regulation of signaling-responsive genes is at the level of transcription. Single-cell quantitative studies of transcription have revealed that transcription occurs intermittently, in bursts. Although the effects of many gene regulatory mechanisms on transcriptional bursting have been studied, it remains unclear how morphogen gradients affect this dynamic property of downstream genes. Here we have adapted smFISH for use in the Drosophila wing imaginal disc in order to measure nascent and mature mRNA of genes downstream of the Wg and Dpp morphogen gradients. We compared our experimental results with predictions from stochastic models of transcription. Our results indicate that the transcription levels of these genes appear to share a common method of control via burst frequency modulation. Our data helps further elucidate the link between developmental gene regulatory mechanisms and transcriptional bursting.

The identification of barely more than 20,000 human genes was amongst the most surprising outcomes of the human genome project. Alternative splicing provides an essential means of expanding the proteome, enabling a single gene to encode multiple, distinct isoforms by selective inclusion or exclusion of exons from mature mRNA. However, mis-regulation of this process is associated with most human diseases. Here, we examine the impact of post-transcriptional processing on extracellular matrix function, focusing on the complex alternative splicing patterns of tenascin-C, a molecule that can exist in as many as 500 different isoforms. We demonstrate that the pro-inflammatory activity of this endogenous innate immune trigger is controlled by inclusion or exclusion of a novel immunomodulatory site located within domains AD2AD1, identifying this as a mechanism that prevents unnecessary inflammation in healthy tissues but enables rapid immune cell mobilization and activation upon tissue damage, and defining how this goes awry in autoimmune disease.

Splicing is the molecular process by which introns are removed from pre-mRNA and exons are joined together to form the sequence of the mature mRNA. While the biochemical steps of splicing have been largely worked out, the ordering and spatial distribution of these steps, especially in relation to the transcriptional process itself, remain controversial. Here, we use single molecule RNA FISH together with expansion microscopy to measure the spatial distribution of nascent and partially spliced transcripts in mammalian cells, allowing us to infer the order in which an intron is transcribed and spliced out of the pre-mRNA. We show that 4 out of 4 genes we interrogated exhibit clear post-transcriptional splicing of at least one intron, and that introns can be spliced in any order. Expansion microscopy further revealed the presence of a transcription site proximal zone in which the motion of RNA is slower than in the nucleoplasm. Full length pre-mRNA undergo continuous splicing as they move through this zone after transcription. Upon leaving this zone, some genes’ transcripts localize to speckles during the process of splicing while others appear to traffic freely through the nucleus without localizing to any other nuclear compartment. Our results suggest a model in which transcription and splicing are largely independent, and the existence of a slow moving zone around the transcription site allows for the unification of co-transcriptional and post-transcriptional models of splicing.

Known as the “fifth RNA nucleotide”, pseudouridine (Ψ or psi) is the first-discovered and most abundant RNA modification occurring at the Uridine site, and it plays a prominent role in a number of biological processes. Thousands of Ψ sites have been identified within different biological contexts thanks to the advancement in high-throughput sequencing technology; nevertheless, the transcriptome-wide distribution, biomolecular functions, regulatory mechanisms, and disease relevance of pseudouridylation are largely elusive. We report here a web server—PIANO—for pseudouridine site (Ψ) identification and functional annotation. PIANO was built upon a high-accuracy predictor that takes advantage of both conventional sequence features and 42 additional genomic features. When tested on six independent datasets generated from four independent Ψ-profiling technologies (Ψ-seq, RBS-seq, Pseudo-seq, and CeU-seq) as benchmarks, PIANO achieved an average AUC of 0.955 and 0.838 under the full transcript and mature mRNA models, respectively, marking a substantial improvement in accuracy compared to the existing in silico Ψ-site prediction methods, i.e., PPUS (0.713 and 0.707), iRNA-PseU (0.713 and 0.712), and PseUI (0.634 and 0.652). Besides, PIANO web server systematically annotates the predicted Ψ sites with post-transcriptional regulatory mechanisms (miRNA-targets, RBP-binding regions, and splicing sites) in its prediction report to help the users explore potential machinery of Ψ. Moreover, a concise query interface was also built for 4,303 known Ψ sites, which is currently the largest collection of experimentally validated human Ψ sites. The PIANO website is freely accessible at: http://piano.rnamd.com.

Physical exercise is universally recognized as stressful. Among the &ldquo, sport species&rdquo, the horse is probably the most appropriate model for investigating the genomic response to stress due to the homogeneity of its genetic background. The aim of this work is to dissect the whole transcription modulation in Peripheral Blood Mononuclear Cells (PBMCs) after exercise with a time course framework focusing on unexplored regions related to introns and intergenic portions. PBMCs NGS from five 3 year old Sardinian Anglo-Arab racehorses collected at rest and after a 2000 m race was performed. Apart from differential gene expression ascertainment between the two time points the complexity of transcription for alternative transcripts was identified. Interestingly, we noted a transcription shift from the coding to the non-coding regions. We further investigated the possible causes of this phenomenon focusing on genomic repeats, using a differential expression approach and finding a strong general up-regulation of repetitive elements such as LINE. Since their modulation is also associated with the &ldquo, exonization&rdquo, the recruitment of repeats that act with regulatory functions, suggesting that there might be an active regulation of this transcriptional shift. Thanks to an innovative bioinformatic approach, our study could represent a model for the transcriptomic investigation of stress.

This review article deals with comprehensive information about the evolutionary history of introns with their localization and functions in the gene transcripts of colorectal cancer precisely. In this way, the major breakthrough in the molecular biology discipline was the discovery of introns by Richard Robert and Phil Sharp in 1977. Firstly, noncoding regions are recognized by various assortments of regulatory ncRNA sequences such as circular RNA, telomere-associated RNA, small nuclear RNA, Piwi-interacting RNA, small interfering RNA, small nucleolar RNA, microRNA, and long noncoding RNA. Fortunately, splicing process of mRNA strand deals with the excision of introns via spliceosomal proteins into mature mRNA which is witnessed only in eukaryotic organisms and devoid of the splicing machinery components in the prokaryotic organisms. The major focal point relies on intronic genes mainly involved in the progression of colorectal cancer with preliminary information. An alternative splicing process takes place in mRNA that implicates in intron retention leading to varied gene expression in cells and tissues and their promotion in colorectal cancer. Therefore, colorectal cancer-associated diseases have paved the way to know more about the intronic genes mainly concentrated among them in the progression of the related diseases. Hence, the focus of the researchers is toward the fascinating cellular and molecular biology aspects of the regulatory intronic sequences known to enhance as well as repress particular gene expression in tumor microenvironment of colorectal cancer by analyzing the genome and proteome levels for the betterment of human kind that is intended for various therapeutic purposes.

Pre-messenger RNA (mRNA) splicing is an essential step in the control of eukaryotic gene expression. During splicing, the introns are removed from the gene transcripts as the exons are ligated to create mature mRNA sequences. Splicing is performed by the spliceosome, which is a macromolecular complex composed of five small nuclear RNAs (snRNAs) and more than 100 proteins. Except for the core snRNP proteins, most spliceosome proteins are transiently associated and presumably involved with the regulation of spliceosome activity. In this study, we explored the association and participation of the human protein RNF113A in splicing. The addition of excess recombinant RNF113A to in vitro splicing reactions results in splicing inhibition. In whole-cell lysates, RNF113A co-immunoprecipitated with U2, U4, and U6 snRNAs, which are components of the tri-snRNP, and with proteins PRP19 and BRR2. When HeLa cells were CRISPR-edited to reduce the RNF113A levels, the in vitro splicing efficiency was severely affected. Consistently, the splicing activity was partially restored after the addition of the recombinant GST-RNF113A. On the basis on these results, we propose a model in which RNF113A associates with the spliceosome by interacting with PRP19, promoting essential rearrangements that lead to splicing.

BackgroundGenetic analysis of BRCA1 and BRCA2 for the diagnosis of hereditary breast and ovarian cancer (HBOC) is commonly restricted to coding regions and exon-intron boundaries. Although germline pathogenic variants in these regions explain about ~20% of HBOC cases, there is still an important fraction that remains undiagnosed. We have screened BRCA1/2 deep intronic regions to identify potential spliceogenic variants that could explain part of the missing HBOC susceptibility.MethodsWe analysed BRCA1/2 deep intronic regions by targeted gene sequencing in 192 high-risk HBOC families testing negative for BRCA1/2 during conventional analysis. Rare variants (MAF ResultsA novel transcript was detected in BRCA1 c.4185+4105C>T variant carrier. This variant promotes the inclusion of a pseudoexon in mature mRNA, generating an aberrant transcript predicted to encode for a non-functional protein. Quantitative and allele-specific assays determined haploinsufficiency in the variant carrier, supporting a pathogenic effect for this variant. Genotyping of 1030 HBOC cases and 327 controls did not identify additional carriers in Spanish population.ConclusionScreening of BRCA1/2 intronic regions has identified the first BRCA1 deep intronic variant associated with HBOC by pseudoexon activation. Although the frequency of deleterious variants in these regions appears to be low, our study highlights the importance of studying non-coding regions and performing comprehensive RNA assays to complement genetic diagnosis.

Precursor-mRNAs (pre-mRNA) can be processed into one or more mature mRNA isoforms through constitutive or alternative splicing pathways. Constitutive splicing of pre-mRNA plays critical roles in gene expressional regulation, such as intron-mediated enhancement (IME), whereas alternative splicing (AS) dramatically increases the protein diversity and gene functional regulation. However, the unavailability of mutants for individual spliced isoforms in plants has been a major limitation in studying the function of mRNA splicing. Here, we describe an efficient tool for manipulating the splicing of plant genes. Using a Cas9-directed base editor, we converted the 5' splice sites in four Arabidopsis genes from the activated GT form to the inactive AT form. Silencing the AS of HAB1.1 (encoding a type 2C phosphatase) validated its function in abscisic acid signaling, while perturbing the AS of RS31A revealed its functional involvement in plant response to genotoxic treatment for the first time. Lastly, altering the constitutive splicing of Act2 via base editing facilitated the analysis of IME. This strategy provides an efficient tool for investigating the function and regulation of gene splicing in plants and other eukaryotes.

Newly synthesized mRNA undergoes several stages of maturation, and the proteins involved in these processes form the mRNP particle. Mature mRNA passes through the nuclear pore from the nucleus to the cytoplasm, where it is transported to the ribosome and mRNA is translated into protein. Recruiting the export and transport factors to the newly synthesized mRNA occurs partly cotranscriptionally. Many of the proteins associate with mRNA from the earliest stages of transcription to its localization in the cytoplasm. Other factors perform their functions strictly at a certain stage. The protein composition of the mRNP particle changes during its adherence to the nuclear pore, passing through it, and being delivered to the site of localization in the cytoplasm. This paper describes the main participants in the export of the mRNP particle, starting with its delivery to the nuclear pore and its attachment to and translocation through the nuclear pore.

Although branchpoint recognition is an essential component of intron excision during the RNA splicing process, the branchpoint itself is frequently assumed to be a basal, rather than regulatory, sequence feature. However, this assumption has not been systematically tested due to the technical difficulty of identifying branchpoints and quantifying their usage. Here, we analyzed ∼1.31 trillion reads from 17,164 RNA sequencing data sets to demonstrate that almost all human introns contain multiple branchpoints. This complexity holds even for constitutive introns, 95% of which contain multiple branchpoints, with an estimated five to six branchpoints per intron. Introns upstream of the highly regulated ultraconserved poison exons of SR genes contain twice as many branchpoints as the genomic average. Approximately three-quarters of constitutive introns exhibit tissue-specific branchpoint usage. In an extreme example, we observed a complete switch in branchpoint usage in the well-studied first intron of HBB (β-globin) in normal bone marrow versus metastatic prostate cancer samples. Our results indicate that the recognition of most introns is unexpectedly complex and tissue-specific and suggest that alternative splicing catalysis typifies the majority of introns even in the absence of differences in the mature mRNA.

RNA splicing is a key process in eukaryotic gene expression, in which an intron is spliced out of a pre-mRNA molecule to eventually produce a mature mRNA. Most intron-containing genes are constitutively spliced, hence efficient splicing of an intron is crucial for efficient regulation of gene expression. Here we use a large synthetic oligo library of ~20,000 variants to explore how different intronic sequence features affect splicing efficiency and mRNA expression levels in S. cerevisiae. Introns are defined by three functional sites, the 5’ donor site, the branch site, and the 3’ acceptor site. Using a combinatorial design of synthetic introns, we demonstrate how non-consensus splice site sequences in each of these sites affect splicing efficiency. We then show that S. cerevisiae splicing machinery tends to select alternative 3’ splice sites downstream of the original site, and we suggest that this tendency created a selective pressure, leading to the avoidance of cryptic splice site motifs near introns’ 3’ ends. We further use natural intronic sequences from other yeast species, whose splicing machineries have diverged to various extents, to show how intron architectures in the various species have been adapted to the organism’s splicing machinery. We suggest that the observed tendency for cryptic splicing is a result of a loss of a specific splicing factor, U2AF1. Lastly, we show that synthetic sequences containing two introns give rise to alternative RNA isoforms in S. cerevisiae, demonstrating that merely a synthetic fusion of two introns might be suffice to facilitate alternative splicing in yeast. Our study reveals novel mechanisms by which introns are shaped in evolution to allow cells to regulate their transcriptome. In addition, it provides a valuable resource to study the regulation of constitutive and alternative splicing in a model organism., Author summary RNA splicing is a process in which parts of a new pre-mRNA are spliced out of the mRNA molecule to produce eventually a mature mRNA. Those RNA segments that are spliced out are termed introns, and they are found in most genes in eukaryotic organisms. Hence regulation of this process has a major role in the control of gene expression. The budding yeast S. cerevisiae is a popular model organism for eukaryotic cell biology, but in terms of splicing it differs, as it has only few intron-containing genes. Nevertheless, this species has been used to study basic principles of splicing regulation based on its ~300 introns. Here we used the technology of a large synthetic genetic library to introduce many new intron-containing genes to the yeast genome, to explore splicing regulation at a wider scope than was possible so far. Reassuringly, our results confirm known regulatory mechanisms, and further expand our understanding of splicing regulation, specifically how the yeast splicing machinery interacts with the end of introns, and how through evolution introns have evolved to avoid unwanted misidentifications of this end. We further demonstrate the potential of the yeast splicing machinery to alternatively splice a two-intron gene, which is common in other eukaryotes but rare in yeast. Our work presents a first-of-its-kind resource for the systematic study of splicing in live cells.

The majority of mammalian genes contain one or more alternative polyadenylation sites. Choice of polyadenylation sites was suggested as one of the underlying mechanisms for generating longer/shorter transcript isoforms. Here, we demonstrate that mature mRNA transcripts can undergo additional cleavage and polyadenylation at a proximal internal site in the 3′-UTR, resulting in two stable, autonomous, RNA fragments: a coding sequence with a shorter 3′-UTR (body) and an uncapped 3′-UTR sequence downstream of the cleavage point (tail). Analyses of the human transcriptome has revealed thousands of such cleavage positions, suggesting a widespread post-transcriptional phenomenon producing thousands of stable 3′-UTR RNA tails that exist alongside their transcripts of origin. By analyzing the impact of microRNAs, we observed a significantly stronger effect for microRNA regulation at the body compared to the tail fragments. Our findings open a variety of future research prospects and call for a new perspective on 3′-UTR-dependent gene regulation., Most mammalian genes contain alternative polyadenylation sites. Here, the authors provide evidence that mRNA can be cleaved post-transcriptionally to generate mRNAs with shorter 3-´UTRs and stable autonomous uncapped 3´-UTR sequences.

Despite highly conserved chromatin states andcis-regulatory elements, studies of metazoan genomes reveal that gene organization and the strategies to control mRNA expression can vary widely among animal species.C. elegansgene regulation is often assumed to be similar to that of other model organisms, yet evidence suggests the existence of distinct molecular mechanisms to pattern the developmental transcriptome, including extensive post-transcriptional RNA control pathways, widespread splice leader (SL) trans-splicing of pre-mRNAs, and the organization of genes into operons. Here, we performed ChIP-seq for histone modifications in highly synchronized embryos cohorts representing three major developmental stages, with the goal of better characterizing whether the dynamic changes in embryonic mRNA expression are accompanied by changes to the chromatin state. We were surprised to find that thousands of promoters are persistently marked by active histone modifications, despite a fundamental restructuring of the transcriptome. We employed global run-on sequencing using a long-read nanopore format to map nascent RNA transcription across embryogenesis, finding that the invariant open chromatin regions are persistently transcribed by Pol II at all stages of embryo development, even though the mature mRNA is not produced. By annotating our nascent RNA sequencing reads into directional transcription units, we find extensive evidence of polycistronic RNA transcription genome-wide, suggesting that nearby genes inC. elegansare linked by shared transcriptional regulatory mechanisms. We present data indicating that the sharing of cis-regulatory sequences has constrainedC. elegansgene positioning and likely explains the remarkable retention of syntenic gene pairs over long evolutionary timescales.

Gene fusions are an important class of mutations in several cancer types and include genomic rearrangements that fuse regulatory or coding elements from two different genes. Analysis of the genetics of cancers harboring fusion oncogenes and the proteins they encode have enhanced cancer diagnosis and in some cases patient treatment. However, the effect of the complex structure of fusion genes on the biogenesis of the resulting chimeric transcripts they express is not well studied. There are two potential RNA‐related vulnerabilities inherent to fusion‐driven cancers: (a) the processing of the fusion precursor messenger RNA (pre‐mRNA) to the mature mRNA and (b) the mature mRNA. In this study, we discuss the effects that the genetic organization of fusion oncogenes has on the generation of translatable mature RNAs and the diversity of fusion transcripts expressed in different cancer subtypes, which can fundamentally influence both tumorigenesis and treatment. We also discuss functional genomic approaches that can be utilized to identify proteins that mediate the processing of fusion pre‐mRNAs. Furthermore, we assert that an enhanced understanding of fusion transcript biogenesis and the diversity of the chimeric RNAs present in fusion‐driven cancers will increase the likelihood of successful application of RNA‐based therapies in this class of tumors. This article is categorized under:RNA Processing > RNA Editing and ModificationRNA Processing > Splicing Regulation/Alternative SplicingRNA in Disease and Development > RNA in Disease, About one‐fifth of cancers harbor a fusion gene associated with disease pathology. The organization of fusion oncogenes can complicate the expression or structure of the mRNAs they express. The dependency of many cancer cells on the efficient biogenesis, and the continued presence of translatable fusion mRNAs offer potential RNA‐related therapeutic vulnerabilities.

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA polymerase II and co-transcriptional capping and splicing of the precursor RNA to form the mature mRNA. During mRNA synthesis, the RNA polymerase II elongation complex is a target for regulation by a large collection of transcription factors that control its catalytic activity, as well as the capping, splicing, and 3'-processing enzymes that create the mature mRNA. Because of the inherent complexity of mRNA synthesis, simpler experimental systems enabling isolation and investigation of its various co-transcriptional stages have great utility. In this article, we describe one such simple experimental system suitable for investigating co-transcriptional RNA capping. This system relies on defined RNA polymerase II elongation complexes assembled from purified polymerase and artificial transcription bubbles. When immobilized via biotinylated DNA, these RNA polymerase II elongation complexes provide an easily manipulable tool for dissecting co-transcriptional RNA capping and mechanisms by which the elongation complex recruits and regulates capping enzyme during co-transcriptional RNA capping. We anticipate this system could be adapted for studying recruitment and/or assembly of proteins or protein complexes with roles in other stages of mRNA maturation coupled to the RNA polymerase II elongation complex.

The hatcing enzyme gene (HE) encodes a protease that is indispensable for the hatching process and is conserved during vertebrate evolution. During teleostean evolution, it is known that HE experienced a drastic transfiguration of gene structure, namely, losing all of its introns. However, these facts are contradiction with each other, since intron-less genes typically lose their original promoter because of duplication via mature mRNA, called retrocopy. Here, using a comparative genomic assay, we showed that HEs have changed their genomic location several times, with the evolutionary timings of these translocations being identical to those of intron-loss. We further showed that HEs maintain the promoter sequence upstream of them after translocation. Therefore, teleostean HEs are unique genes which have changed intra- (exon-intron) and extra-genomic structure (genomic loci) several times, although their indispensability for the reproductive process of hatching implies that HE genes are translocated by retrocopy with their promoter sequence.