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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.

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.

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.

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.

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.

The process of removing intronic sequences from a precursor to messenger RNA (pre-mRNA) to yield a mature mRNA transcript via splicing is an integral step in eukaryotic gene expression. Splicing is carried out by a cellular nanomachine called the spliceosome that is composed of RNA components and dozens of proteins. Despite decades of study, many fundamentals of spliceosome function have remained elusive. Recent developments in single-molecule fluorescence microscopy have afforded new tools to better probe the spliceosome and the complex, dynamic process of splicing by direct observation of single molecules. These cutting-edge technologies enable investigators to monitor the dynamics of specific splicing components, whole spliceosomes, and even cotranscriptional splicing within living cells. WIREs RNA 2016, 7:683-701. doi: 10.1002/wrna.1358 For further resources related to this article, please visit the WIREs website.

Eukaryotic precursor-messenger RNAs (pre-mRNAs) undergo splicing to remove intragenic regions (introns) and ligate expressed regions (exons) together. Unlike exons in the mature messenger RNAs (mRNAs) that are used for translation, introns that are spliced out of pre-mRNAs were generally believed to lack function and to be degraded. However, recent studies have revealed that a large group of spliced introns can escape complete degradation and are processed to generate noncoding RNAs (ncRNAs), including different types of small RNAs, long-noncoding RNAs, and circular RNAs. Strikingly, exonic sequences can be also back-spliced from pre-mRNAs to form stable circular RNAs. Together, the findings that ncRNAs can be spliced out of mRNA precursors not only expand the ever-growing repertoire of ncRNAs that originate from different genomic regions, but also reveal the unexpected transcriptomic complexity and functional capacity of eukaryotic genomes.

In mammalian cells in general and in neurons in particular, mRNA maturation, translation, and degradation are highly complex and dynamic processes. RNA-binding proteins (RBPs) play crucial roles in all these events. First, they participate in the choice of pre-mRNA splice sites and in the selection of the polyadenylation sites, determining which of the possible isoforms is produced from a given precursor mRNA. Then, once in the cytoplasm, the protein composition of the RNP particles determines whether the mature mRNA is transported along the dendrites or the axon of a neuron to the synapses, how efficiently it is translated, and how stable it is. In agreement with their importance for neuronal function, mutations in genes that code for RBPs are associated with various neurological diseases. In this review, we illustrate how individual RBPs determine the fate of an mRNA, and we discuss how mutations in RBPs or perturbations of the mRNA metabolism can cause neurodegenerative disorders.

Structured RNA elements within messenger RNA often direct or modulate the cellular production of active proteins. As reviewed here, RNA structures have been discovered that govern nearly every step in protein production: mRNA production and stability; translation initiation, elongation, and termination; protein folding; and cellular localization. Regulatory RNA elements are common within RNAs from every domain of life. This growing body of RNA-mediated mechanisms continues to reveal new ways in which mRNA structure regulates translation. We integrate examples from several different classes of RNA structure-mediated regulation to present a global perspective that suggests that the secondary and tertiary structure of RNA ultimately constitutes an additional level of the genetic code that both guides and regulates protein biosynthesis.

Endogenes rarely support transitive silencing, whereas most transgenes generally allow the spread of silencing to occur along the primary target. To determine whether the presence of introns might explain the difference, we investigated the influence of introns in the primary target on 3'–5' silencing transitivity. When present in a transgene, an intron-containing endogene fragment does not prohibit the spread of silencing across this fragment, indicating that introns do not preclude silencing transitivity along endogenes. Also, a multiple intron-containing genomic gene fragment that had previously been shown not to support transitivity in an endogenous context could support transitivity when present in a transgene. Nevertheless, genomic intron-containing fragments delayed the onset and diminished the efficiency of transitive silencing of a secondary target compared with the corresponding cDNA fragments. Remarkably, transitivity was impaired proportionally with the length of the pre-mRNA, and not of the mRNA. The latter result suggests that the RNA dependent RNA polymerase-based spreading of silencing progresses along the non-spliced rather than the fully processed mature mRNA.

Full-length retroviral RNA has three well-established functions: it constitutes the genomic RNA that is packaged into virions and is transmitted to target cells by infection, it is the messenger RNA (mRNA) template for viral Gag and Pol protein synthesis and it serves as the pre-mRNA for the production of subgenomic spliced mRNAs that encode additional viral proteins such as Env. More recent work indicates that these full-length RNAs also play important roles in the assembly of virus particles, not only as a structural scaffold that facilitates viral core formation but also as a potential regulator of the assembly process itself. Here, we discuss how these assorted activities may be coupled with each other, paying particular attention to the importance of RNA trafficking and subcellular localization in the cytoplasm, possible points of regulation, and the role(s) played by cellular RNA-binding proteins.

Direct localization of specific genes, RNAs, and proteins has allowed the dissection of individual nuclear speckles in relation to the molecular biology of gene expression. Nuclear speckles (aka SC35 domains) are essentially ubiquitous structures enriched for most pre-mRNA metabolic factors, yet their relationship to gene expression has been poorly understood. Analyses of specific genes and their spliced or mature mRNA strongly support that SC35 domains are hubs of activity, not stores of inert factors detached from gene expression. We propose that SC35 domains are hubs that spatially link expression of specific pre-mRNAs to rapid recycling of copious RNA metabolic complexes, thereby facilitating expression of many highly active genes. In addition to increasing the efficiency of each step, sequential steps in gene expression are structurally integrated at each SC35 domain, consistent with other evidence that the biochemical machineries for transcription, splicing, and mRNA export are coupled. Transcription and splicing are subcompartmentalized at the periphery, with largely spliced mRNA entering the domain prior to export. In addition, new findings presented here begin to illuminate the structural underpinnings of a speckle by defining specific perturbations of phosphorylation that promote disassembly or assembly of an SC35 domain in relation to other components. Results thus far are consistent with the SC35 spliceosome assembly factor as an integral structural component. Conditions that disperse SC35 also disperse poly(A) RNA, whereas the splicing factor ASF/SF2 can be dispersed under conditions in which SC35 or SRm300 remain as intact components of a core domain.

Background Duchenne muscular dystrophy (DMD) is an X-linked recessive muscle wasting disorder characterised by the absence of the protein dystrophin. Antisense oligonucleotides have been used to re-direct dystrophin pre-mRNA processing by blocking sequences crucial to pre-mRNA splicing, thereby inducing skipping of specific exons. We wished to determine which splicing motifs are most amenable as targets for antisense oligonucleotide induction of efficient and specific skipping of selected exons. Methods Antisense oligonucleotides were directed at regions of dystrophin exon 19 involved in pre-mRNA splicing, including the donor and acceptor splice sites and the exon splicing enhancer (ESE). Cultured myotubes were transfected with antisense oligonucleotides at various concentrations and studies undertaken to determine both specificity and efficiency of induced exon 19 skipping. Results Antisense oligonucleotides as small as 12 nucleotides targeting the ESE induced consistent and specific skipping of only exon 19 in both human and normal and mdx mouse myotubes. Antisense oligonucleotides directed at the donor and acceptor splice sites also induced specific exon 19 skipping while mismatched antisense oligonucleotides could only induce skipping when delivered at higher concentrations. No other dystrophin exons were removed from the mature mRNA as a consequence of these antisense oligonucleotides treatments. Conclusions Antisense oligonucleotides directed at the ESE tended to be marginally more efficient than those which targeted the donor or acceptor splice sites, based on their ability to induce specific skipping at lower concentrations. The specificity of exon removal does not appear to be a function of target selection, but may reflect the combination of the splicing motifs and position of that exon in the pre-mRNA.

Covalent RNA modifications were recently rediscovered as abundant RNA chemical tags. Similarly to DNA epigenetic modifications, they have been proposed as essential regulators of gene expression. Here we focus on 3 of the most abundant adenosine methylations: N6-methyladenosine (m6 A), N6,2'-O-dimethyladenosine (m6 Am) and N1-methyladenosine (m1 A). We review the potential role of these modifications on mature mRNA in regulating gene expression within the adult brain, nervous system function and normal and pathological behavior. Dynamic mRNA modifications, summarized as the epitranscriptome, regulate transcript maturation, translation and decay, and thus crucially determine gene expression beyond primary transcription regulation. However, the extent of this regulation in the healthy and maladapted adult brain is poorly understood. Analyzing this novel layer of gene expression control in addition to epigenetics and posttranslational regulation of proteins will be highly relevant for understanding the molecular underpinnings of behavior and psychiatric disorders.

The expression of protein-encoding genes is a complex process culminating in the production of mature mRNA and its translation by the ribosomes. The production of a mature mRNA involves an intricate series of processing steps. The majority of eukaryotic protein-encoding genes contain intron sequences that disrupt the protein-encoding frame, and hence have to be removed from immature mRNA prior to translation into protein. The mechanism involved in the selection of correct splice sites is incompletely understood. A considerable body of evidence suggests that the splicing machinery has suboptimal efficiency and fidelity leading to substantial processing inaccuracy. Here we discuss a recently published article that extends observations that cells rely on nonsense-mediated mRNA decay (NMD) to compensate for such suboptimal processing accuracy. Intriguingly these authors provide evidence for a strong selective pressure in favour of premature termination of mRNA translation in the event of intron retention. The analysis presented implies a positive role of NMD in transcript diversification through alternative splicing and suggest that this ancient surveillance mechanism may have co-evolved with intron acquisition born from the need for quality control of splicing patterns.

We have identified and analyzed a Drosophila melanogaster gene that encodes a chloride channel subunit (DrosGluCl-α) previously shown to function as a glutamategated chloride channel in an in vitro expression system. Sequence analysis of several cDNAs corresponding to the gene revealed sequence diversity in their open reading frames at seven specific sites. Site-specific A-to-G variations between cDNA and genomic sequences, consistent with RNA editing, were detected at five nucleotide positions. In addition, sequence variations among cDNA clones consistent with alternative splicing of mRNA were found at two different sites. In the 5′ region, two small adjacent exons, containing similar but distinct modular sequences, are alternatively incorporated into the mature mRNA. In the 3′ region, alternative splicing generates a variant encoding a protein with four additional amino acids just upstream of the fourth transmembrane domain. Combinations of RNA editing and alternative splicing can lead to extensive diversification of transcripts. These results give the first example of RNA editing in neurotransmitter-gated chloride channel genes or of alternative splicing in a glutamate-gated chloride channel gene of Drosophila.

Premature termination codons (PTCs) can cause the decay of mRNAs in the nuclear fraction of mammalian cells. This enigmatic nuclear response is of interest because it suggests that translation signals do not restrict their effect to the cytoplasm, where fully assembled ribosomes reside. Here we examined the molecular mechanism for this putative nuclear response by using the T-cell receptor-beta (TCR-beta) gene, which acquires PTCs as a result of programmed rearrangements that occur during normal thymic ontogeny. We found that PTCs had little or no measurable effect on TCR-beta pre-mRNA levels, but they sharply depressed TCR-beta mature mRNA levels in the nuclear fraction of stably transfected cells. A PTC split by an intron was able to trigger the down-regulatory response, implying that PTC recognition occurs after an mRNA is at least partially spliced. However, intron deletion and addition studies demonstrated that a PTC must be followed by at least one functional (spliceable) intron to depress mRNA levels. One explanation for this downstream intron-dependence is that cytoplasmic ribosomes adjacent to nuclear pores scan mRNAs still undergoing splicing as they emerge from the nucleus. We found this explanation to be unlikely because PTCs only 8 or 10 nt upstream of a terminal intron down-regulated mRNA levels, even though this distance is too short to permit PTC recognition in the cytoplasm prior to the splicing of the downstream intron in the nucleus. Collectively, the results suggest that nonsense codon recognition may occur in the nucleus.

1. The type-1 angiotensin II (AngII) receptors, designated AT(1), mediate most of the biological actions of the peptide hormone AngII. They are the most recent drug target for the treatment of hypertension and cardiac failure and basic research is now focusing on the mechanisms that regulate their expression. 2. In humans there is a single AT(1) gene. It encodes a 47 kb pre-mRNA containing five exons, with the previously described AT(1) open reading frame (ORF) on exon 5. Alternative splicing results in the production of mature mRNA that are translated at different efficiencies and encode two receptor isoforms. The inclusion of exon 2 markedly inhibits translation of the downstream ORF, both in vitro and in vivo. Nonetheless, this exon is present in up to one-half of AT(1) mRNA in all tissues studied. 3. Transcripts containing exon 3 spliced to exon 5 encode a receptor with an amino-terminal extension of 32 amino acids and represent up to one-third of total AT(1) mRNA in each tissue examined. In vitro, these latter transcripts are translated to produce a longer receptor and, in transfected cells, they encode a functional AT(1) receptor with ligand-binding and signalling properties similar to those of the short isoform. 4. Exon 4 is of minor significance as it is rarely spliced into AT(1) mRNA. 5. These data indicate that, in addition to characterizing factors that modulate AT(1) promoter activity and RNA stability, it is important to analyse the splicing patterns of this gene when studying the regulation of its expression.

Messenger RNA maturation in eukaryotes typically involves the removal of introns from long precursor molecules. An unusual form of RNA splicing in which separate precursor transcripts contribute sequences to the mature mRNA through intermolecular reactions has now been documented in a number of diverse organisms. In this review, the phenomenon of pre-mRNA trans-splicing has been divided into two categories. The "spliced leader" type, found in protozoans such as trypanosomes and lower invertebrates such as nematodes, results in the addition of a short, capped 5' noncoding sequence to the mRNA. The "discontinuous group II intron" form of trans-splicing, found in plant/algal chloroplasts and plant mitochondria, involves the joining of independently transcribed coding sequences, presumably through interactions between "intronic" RNA pieces. Both categories of trans-splicing are mechanistically similar to conventional nuclear pre-mRNA cis-splicing; potential evolutionary relationships are discussed.

mRNA‐splicing presents an important regulatory step that contributes to the functional repertoire of all cell types and tissues. Profound changes in alternative splicing have also been revealed during embryonic stem cell (ESC) differentiation. Based on a genome‐wide screen for pluripotency regulators, Ng and colleagues now identify SON as a new splicing regulator in the maintenance of human ESC pluripotency and self‐renewal (Lu et al, 2013). RNA splicing is the process of removal of introns from nascent pre‐mRNAs that results in the formation of mature mRNAs, with all exons joined together. The human genome contains ∼40 000 genes that give rise to ∼200 000 transcripts, suggesting that each gene locus can generate several different transcripts. This phenomenon can be in part explained by alternative splicing. Alternative splicing occurs when an exon can be either included or excluded from the mature mRNA, giving rise to different transcripts called splice variants. Transcriptome analysis suggests that the vast majority of human multiexonic genes are alternatively spliced (Pan et al, 2008), and this occurs in a cell‐type‐specific …

The recruitment and binding of a messenger RNA (mRNA) to the ribosome is a highly specific and regulated process. This process depends on unique sequences and structures within the mRNA, soluble auxiliary factors as well as the RNA and protein components of the ribosome to allow the synthesis of proteins. Keywords: ribosome; messenger RNA; IRES; translation factor; Shine–Dalgarno

The effect of tRNA distribution in the cell on the rate of specific protein synthesis has been investigated. A tRNA-dependent cell-free protein-synthesizing system derived from Krebs-II ascites cells has been worked out. In this system it is possible to synthesize specific proteins (egg-white proteins or globin) by the translation of oviduct or reticulocyte messenger RNA in the presence of tRNA from homologous or heterologous tissues. The rate of translation of a given messenger RNA is optimal in the presence of tRNA from the homologous tissue. The lower level of protein synthesis obtained with an excess of heterologous tRNA can be overcome by adding the homologous tRNA. Nevertheless homologous isoaccepting species partly purified by a reversed-phase chromatography or a benzoylated DEAE-cellulose column cannot fully restore the optimal synthesis, eliminating the hypothesis according to which a particular tRNA species is involved in this phenomenon. The correct distribution of tRNA is necessary for optimal translation of a messenger RNA.

Different proteins are secreted by the various regions of rat epididymis. We have examined the messenger RNA dependence of this varied gene expression by cell-free translation of poly(A)RNA extracts from initial segment, caput plus corpus, and cauda. Labeled translation products were analyzed by polyacrylamide gel electrophoresis under denaturing conditions. Poly(A)RNA from initial segment coded for several unique bands of labeled protein, including a 23,000 MW protein that may correspond to an initial segment protein reported previously to be regulated by testicular fluid factors. Messenger RNA encoding 20,000 MW protein believed to be alpha-lactalbumin, was most abundant in caput but also present in initial segment. Acidic epididymal glycoprotein (AEG) was identified previously by immunoperoxidase staining in epithelial cells of caput, corpus, and cauda. AEG was not readily identified on electrophoresis of total translated proteins, but when concentrated by immunoprecipitation with purified AEG antibody prior to electrophoresis, AEG appeared in both caput plus corpus, and in cauda poly(A)RNA translations.

Mouse globin messenger RNA has been degraded by ribonuclease T1 to give poly(adenylic acid) fragments of two size classes which run electrophoretically with standard RNA molecules approximately 75 and 50 nucleotides long. These contain no internal ribonucleotides other than adenosine and occur at the 3′-end of the mRNA molecule. The probable sequence to the 5′-side of the poly(A) tract is -G-C. Attempts to separate the α and β mRNA activities by formamide gradient elution from poly(U)-Sepharose were unsuccessful. The evidence is more compatible with a post-transcriptional end-addition mechanism for the synthesis of the poly(adenylic acid) region of messenger RNA than a transcriptional mechanism.

There are gaps in what is known about the metabolism of some mammalian small RNA species. Our present observations can be summarized as follows. The level of radiolabeled mature U1 RNA doubled between 2 and 24 hr of label chase, while that of all other small RNA species tested did not change. These results are compatible with the possibility that the nucleotide precursor pool for U1 RNA transcription may be partly segregated, or that there may be a second pathway of U1 RNA formation. Precursors of nucleolar U3 RNA were detected whose electrophoretic mobilities are equivalent to those of transcripts approximately 14 and approximately 8 nucleotides longer than the mature species, and which are apparently cytoplasmic. The ladder of U2 RNA precursors showed a gap, suggesting that some of the cleavages during U2 RNA processing are endonucleolytic. We detected an apparent U5 RNA precursor whose electrophoretic mobility is equivalent to that of a species approximately 1 nucleotide longer than mature U5 RNA. There was a predominant band in the middle of the ladder of U4 RNA precursors (apparently approximately 3 nucleotides longer than mature U4 RNA) which suggests that U4 RNA maturation may pause briefly at that intermediate. There are apparently two additional species of mature hY3 RNA, which are less abundant and are about 1 and 2 bases longer than the major hY3 RNA species. An apparent hY3 RNA precursor was detected, which may be approximately 2-3 nucleotides longer than the main mature hY3 RNA species.

Equilibrium studies of the interaction between messenger RNA and the rabbit reticulocyte initiation factor that binds methionyl-tRNAf, eIF-2, were performed, using filtration through nitrocellulose membranes to measure complex formation. The formation of complexes between the initiation factor and messenger RNA is shown to be consistent with a simple, bimolecular equilibrium. Binding of native, translatable 125I-labeled globin messenger RNA to the factor is tight, with an apparent dissociation constant of approximately 5 × 10 −10 M at physiological salt concentration. This binding does not involve the poly(A) sequence at the 3′ end of globin messenger RNA, nor the 90-nucleotide sequence adjacent to it, for poly(A)-free derivatives of globin messenger RNA prepared by controlled, processive phosphorolysis bind the initiation factor with an affinity essentially equal to that of native globin messenger RNA. In contrast to other cases of protein-nucleic-acid interaction, binding of initiation factor eIF-2 to messenger RNA is as tight at 0.15 M KCI as it is at 0.01 M KCI and is consistent with a preference for the RNA conformation existing at physiological salt concentration. 32P-labeled bacteriophage R17 RNA, a messenger RNA that can be translated, although at relatively low efficiency, in a mammalian cell-free system, also forms an equimolar complex with eIF-2, but binds 10 to 15-fold less tightly than globin messenger RNA. These results show that the eukaryotic initiation factor that binds methionyl-tRNAf recognizes a sequence in globin messenger RNA that does not include the poly(A) moiety or most of the untranslated sequence at the 3′ end of the molecule. The data suggest a relationship between the affinity of a given messenger RNA species for initiation factor eIF-2 and its efficiency as a template in protein synthesis.

The initiation site for transcripotion of the 30-kDa protein mRNA of tobacco mosaic virus was mapped uniquely at residue 1558 from the 3'-terminus on TMV RNA using the primer-extension and the S1-nuclease mapping method.

Hen oviduct nuclear matrix-bound mature ovalbumin mRNA is released from the matrix in the presence of ATP, while the ovalbumin mRNA precursors remain bound to this structure. Detachment of the mature mRNA from the matrix by ATP as well as ATP-dependent efflux of mRNA from isolated nuclei were found to be inhibited by cytochalasin B. On the other hand, in the absence of ATP, cytochalasin B exclusively caused the release (and nucleocytoplasmic efflux) of the ovalbumin messenger precursors, but not of the mature mRNA. After cytochalasin B treatment, actin could be detected in the matrix supernatant. Phalloidin which stabilizes actin filaments did not cause RNA liberation in the absence of ATP, but inhibited the ATP-induced detachment of mature mRNA. RNA release was also achieved with a monoclonal antibody against actin but not with monoclonal antibodies against tubulin and intermediate filaments. These results suggest that actin-containing filaments are involved in the restriction of immature messengers to the cell nucleus.

Messenger RNA has been isolated from rat lens tissue. The mRNA species which codes for the A2 chain of alpha-crystallin, revealed the same extremely high sedimentation value (14S) as the corresponding messenger from calf lens. However, it has been shown that in rat lens the 14-S messenger preparation directs the synthesis of an additional alpha-crystallin chain, designated as alpha-X with an approximate molecular weight of 24000. For comparative purpose crystallin synthesis has also been studied as well in cultured rat lens cells as in the rat lens cell-free system.

The effect of modification of the secondary structure of phage f2 RNA and brome mosaic virus (BMV) RNA 3 on the formation of initiation complexes in Escherichia coli and wheat germ protein-synthesizing systems was studied. Modification of the RNAs was achieved by using O-methylhydroxylamine, which specifically reacts with cytosines; this leaves the initiation codons unchanged and, under denaturing conditions, leads to irreversible unfolding of the RNA. E. coli ribosomes interact with newly exposed AUG/GUG codons in the modified templates forming polysomes, whereas they form monosomes with native f2 RNA or BMV RNA 3. With wheat germ ribosomes, disomes are formed in the presence of BMV RNA 3, either native or modified. With f2 RNA, eukaryotic ribosomes form monosomes, independent of the secondary structure of the template. The results indicate that, in contrast to prokaryotic ribosomes, binding of eukaryotic ribosomes to f2 RNA or BMV RNA 3 is not affected by modification of the secondary structure of these messengers.

A novel interaction between the 5′-untranslated region of eukaryotic messenger RNAs and non-contiguous sequences in the 18 S ribosomal RNA is proposed. The small ribosomal RNA contains, at its 3′-terminus, a heavily conserved hairpin structure. It is suggested that mRNA 5′-leader sequence stabilises this structure by interacting with other conserved nucleotides which flank it. Sequences closely related to the required sequence (A—U—C—C—A—C—C) occur quite commonly in eukaryotic mRNAs and are often found immediately upstream from the AUG-codon. This interaction may have a role in the events which lead up to the initiation of protein synthesis.

There is evidence that the polyribosomal messenger RNA (mRNA) in animal cells is specifically associated with proteins [l-7] . In the case of cytoplasmic [5,8] or nuclear [9] messenger-like RNA (mlRNA) similar RNA-protein (RNP) complexes have also been described. Contrary to the claim that such associations may be non-specific [ 10, 1 l] , adequate control experiments suggest that they pre-exist in the cell and are not artefacts produced during lysis [5,8, 121. The exact physiological significance of these structures is not known. They may play a variety of roles such as: protection against RNase attack [5,8], RNA transport [ 1,8,9] , cleavage of giant mlRNA and selection of the RNA molecules to be transferred to the cytoplasm [5,9] , or initiation of protein synthesis [7] . In particular, two reports [ 13, 141 claim that the protein bound to the giant nuclear mlRNA is the same as that associated with mRNA in polyribosomes, indicating a possible function in mRNA transport. We have studied these mRNP particles in a highly differentiated system: duck immature red blood cells. In contrast to the hemoglobin producing cells in mammals, duck red cells are nucleated, making possible the investigation of the mland mRNA complexes in the nucleus and in the cytoplasm of a cell where a specific mRNA can be identified.

Phage M12 RNA, serving as a messenger for protein synthesis in vitro, is incorporated into polysomal structures containing up to ten ribosomes per RNA molecule. If this RNA messenger is repressed by M12 coat protein prior to its translation, it binds a maximum of four ribosomes per RNA genome. This difference in polysome distribution is similar to the size difference between phage specific polysomes formed at early and late times of viral development in the infected cell. Such a finding indicates that the theory of translational repression, as it has been proposed from previous experiments in vitro, is also a major control mechanism of phage protein synthesis in vivo.