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Mitochondria are cellular organelles critical for numerous cellular processes and harboring their own circular mitochondrial DNA (mtDNA). Most mtDNA associated disorders (either deletions, mutations, or depletion) lead to multisystemic disease, often severe at a young age, with no disease-modifying therapies. Mitochondria have a capacity to enter eukaryotic cells and to be transported between cells. We describe a method of ex vivo augmentation of hematopoietic stem and progenitor cells (HSPCs) with normal exogenous mitochondria, termed mitochondrial augmentation therapy (MAT). Here, we show that MAT is feasible and dose dependent, and improves mitochondrial content and oxygen consumption of healthy and diseased HSPCs. Ex vivo mitochondrial augmentation of HSPCs from a patient with a mtDNA disorder leads to superior human engraftment in a non-conditioned NSGS mouse model. Using a syngeneic mouse model of accumulating mitochondrial dysfunction (Polg), we show durable engraftment in non-conditioned animals, with in vivo transfer of mitochondria to recipient hematopoietic cells. Taken together, this study supports MAT as a potential disease-modifying therapy for mtDNA disorders.

Hepatitis C virus (HCV) infection is the leading cause of chronic hepatitis, which often results in liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). HCV possesses an RNA genome and its replication is confined to the cytoplasm. Yet, infection with HCV leads to global changes in gene expression, and chromosomal instability (CIN) in the host cell. The mechanisms by which the cytoplasmic virus affects these nuclear processes are elusive. Here, we show that HCV modulates the function of the Structural Maintenance of Chromosome (SMC) protein complex, cohesin, which tethers remote regions of chromatin. We demonstrate that infection of hepatoma cells with HCV leads to up regulation of the expression of the RAD21 cohesin subunit and changes cohesin residency on the chromatin. These changes regulate the expression of genes associated with virus-induced pathways. Furthermore, siRNA downregulation of viral-induced RAD21 reduces HCV infection. During mitosis, HCV infection induces hypercondensation of chromosomes and the appearance of multi-centrosomes. We provide evidence that the underlying mechanism involves the viral NS3/4 protease and the cohesin regulator, WAPL. Altogether, our results provide the first evidence that HCV induces changes in gene expression and chromosome structure of infected cells by modulating cohesin.

Mutation data reveal the dynamic equilibrium between DNA damage and repair processes in cells and are indispensable to the understanding of age-related diseases, tumor evolution, and the acquisition of drug resistance. However, available genome-wide methods have a limited ability to resolve rare somatic variants and the relationships between these variants. Here, we present lineage sequencing, a new genome sequencing approach that enables somatic event reconstruction by providing quality somatic mutation call sets with resolution as high as the single-cell level in subject lineages. Lineage sequencing entails sampling single cells from a population and sequencing subclonal sample sets derived from these cells such that knowledge of relationships among the cells can be used to jointly call variants across the sample set. This approach integrates data from multiple sequence libraries to support each variant and precisely assigns mutations to lineage segments. We applied lineage sequencing to a human colon cancer cell line with a DNA polymerase epsilon (POLE) proofreading deficiency (HT115) and a human retinal epithelial cell line immortalized by constitutive telomerase expression (RPE1). Cells were cultured under continuous observation to link observed single-cell phenotypes with single-cell mutation data. The high sensitivity, specificity, and resolution of the data provide a unique opportunity for quantitative analysis of variation in mutation rate, spectrum, and correlations among variants. Our data show that mutations arrive with nonuniform probability across sublineages and that DNA lesion dynamics may cause strong correlations between certain mutations.

Splicing and alternative splicing of pre-mRNA are key sources in the formation of diversity in the human proteome. These processes have a central role in the regulation of the gene expression pathway. Yet, how spliceosomes are assembled on a multi-intronic pre-mRNA is at present not well understood. To study the spliceosomes assembled in vivo on transcripts with variable number of introns, we examined a series of three related transcripts derived from the β-globin gene, where two transcript types contained increasing number of introns, while one had only an exon. Each transcript had multiple MS2 sequence repeats that can be bound by the MS2 coat protein. Using our protocol for isolation of endogenous spliceosomes under native conditions from cell nuclei, we show that all three transcripts are found in supraspliceosomes – 21 MDa dynamic complexes, sedimenting at 200S in glycerol gradients, and composed of four native spliceosomes connected by the transcript. Affinity purification of complexes assembled on the transcript with most introns (termed E6), using the MS2 tag, confirmed the assembly of E6 in supraspliceosomes with components such as Sm proteins and PSF. Furthermore, splicing inhibition by spliceostatin A did not inhibit the assembly of supraspliceosomes on the E6 transcript, yet increased the percentage of E6 pre-mRNA supraspliceosomes. These findings were corroborated in intact cells, using RNA FISH to detect the MS2-tagged E6 mRNA, together with GFP-tagged splicing factors, showing the assembly of splicing factors SRSF2, U1-70K, and PRP8 onto the E6 transcripts under normal conditions and also when splicing was inhibited. This study shows that different transcripts with different number of introns, or lacking an intron, are assembled in supraspliceosomes even when splicing is inhibited. This assembly starts at the site of transcription and can continue during the life of the transcript in the nucleoplasm. This study further confirms the dynamic and universal nature of supraspliceosomes that package RNA polymerase II transcribed pre-mRNAs into complexes composed of four native spliceosomes connected by the transcript, independent of their length, number of introns, or splicing state.

Existing cancer benchmark data sets for human sequencing data use germline variants, synthetic methods, or expensive validations, none of which are satisfactory for providing a large collection of true somatic variation across a whole genome. Here we propose a data set, Lineage derived Somatic Truth (LinST), of short somatic mutations in the HT115 colon cancer cell-line, that are validated using a known cell lineage that includes thousands of mutations and a high confidence region covering 2.7 gigabases per sample., Megan Shand et al. present Lineage derived Somatic Truth (LinST), a validated data set of somatic mutations from a colon cancer cell line with a known lineage tree structure. They show that LinST can be used to benchmark true-positive and false-positive rates in somatic variant-calling pipelines applied to cancer genomic data.

Gene expression dynamics can be measured in single living cells. Using a detectable transcriptionally active gene in living cells, we previously found that an mRNA undergoing several splicing events was retained at this gene after transcription until completion of mRNA processing. To determine the reason for this delay in release and whether mRNA retention on the gene might depend on splicing factor availability, we modulated the levels of splicing factors in the nucleus. Increasing the abundance of the diffusing fraction of splicing factors by their overexpression or by Clk1 kinase overexpression to disassemble nuclear speckles, led to a reduction in splicing factor residence times on the active gene, and the retained mRNA was rapidly released from the gene. Other treatments such as overexpression of a mutant inactive Clk1, the downregulation of MALAT1 lncRNA or of the Son protein, or the overexpression of the splicing factor import factor TNPO3, did not affect the dynamics of mRNA release from the gene. We found that the faster release of the mRNA from the gene mediated by increased availability of splicing factors, was dependent on the RS domain of the splicing factors and its phosphorylation state. We propose that the relative abundancies of splicing factors in the nucleoplasm can affect their availability for the splicing events taking place, and regulate the kinetics of mRNA release from the gene after processing., Author summary Genetic information is contained in the cell nucleus and encodes proteins. However, protein production takes place in the cytoplasm, and so a molecule is needed to connect between the nucleus and cytoplasm. This messenger molecule is called messenger RNA (mRNA). It is produced and copied from the DNA, and after some processing will travel to the cytoplasm to encode proteins. This study focuses on the timing of mRNA release from the gene after it is copied from the DNA. Processing of mRNA includes the removal of some of its pieces and the stitching back of the remaining parts. This is called splicing. We found that mRNAs undergoing many splicing events are retained on the gene until splicing has completed, and examined what is the cause for this delay. We found that the factors performing the splicing might be limiting the process if their levels are not high enough at the gene locus. By increasing splicing factor levels in the nucleus we show that their abundance increases the rate at which mRNA is released. This means that the cell can regulate gene expression rates by limiting the availability of splicing factors that are free to take part in the processing of mRNA.

Microsatellites (MSs) are tracts of variable-length repeats of short DNA motifs that exhibit high rates of mutation in the form of insertions or deletions (indels) of the repeated motif. Despite their prevalence, the contribution of somatic MS indels to cancer has been largely unexplored, owing to difficulties in detecting them in short-read sequencing data. Here we present two tools: MSMuTect, for accurate detection of somatic MS indels, and MSMutSig, for identification of genes containing MS indels at a higher frequency than expected by chance. Applying MSMuTect to whole-exome data from 6, 747 human tumors representing 20 tumor types, we identified >1, 000 previously undescribed MS indels in cancer genes. Additionally, we demonstrate that the number and pattern of MS indels can accurately distinguish microsatellite-stable tumors from tumors with microsatellite instability, thus potentially improving classification of clinically relevant subgroups. Finally, we identified seven MS indel driver hotspots: four in known cancer genes (ACVR2A, RNF43, JAK1, and MSH3) and three in genes not previously implicated as cancer drivers (ESRP1, PRDM2, and DOCK3).

The transcriptional kinetics of RNA polymerase II, the enzyme responsible for mRNA transcription in the nucleoplasm, can be modulated by a variety of factors. It is therefore important to establish experimental systems that will enable the readout of transcription kinetics of specific genes as they occur in real time within individual cells. This can be performed by implementing fluorescent tagging of the mRNA under live-cell conditions. This chapter describes how to generate fluorescently tagged genes and mRNA, and how a photobleaching approach can produce information on mRNA transcription kinetics.

Existing somatic benchmark datasets for human sequencing data use germline variants, synthetic methods, or expensive validations, none of which are satisfactory for providing a large collection of true somatic variation across a whole genome. Here we propose a dataset of short somatic mutations, that are validated using a known cell lineage. The dataset contains 56,974 (2,687 unique) Single Nucleotide Variations (SNV), 6,370 (316 unique) small Insertions and Deletions (Indels), and 144 (8 unique) Copy Number Variants (CNV) across 98 in silico mixed truth sets with a high confidence region covering 2.7 gigabases per mixture. The data is publicly available for use as a benchmarking dataset for somatic short mutation discovery pipelines.

Splicing can occur co-transcriptionally. What happens when the splicing reaction lags after the completed transcriptional process? We found that elongation rates are independent of ongoing splicing on the examined genes and suggest that when transcription has completed but splicing has not, the splicing machinery is retained at the site of transcription, independently of the polymerase.

Nuclear transcribed genes produce mRNA transcripts destined to travel from the site of transcription to the cytoplasm for protein translation. Certain transcripts can be further localized to specific cytoplasmic regions. We examined the life cycle of a transcribed β-actin mRNA throughout gene expression and localization, in a cell system that allows the in vivo detection of the gene locus, the transcribed mRNAs and the cytoplasmic β-actin protein that integrates into the actin cytoskeleton. Quantification showed that RNA polymerase II elongation progressed at a rate of 3.3 kb/minute and that transactivator binding to the promoter was transient (40 seconds), and demonstrated the unique spatial structure of the coding and non-coding regions of the integrated gene within the transcription site. The rates of gene induction were measured during interphase and after mitosis, demonstrating that daughter cells were not synchronized in respect to transcription initiation of the studied gene. Comparison of the spatial and temporal kinetics of nucleoplasmic and cytoplasmic mRNA transport showed that the β-actin-localization response initiates from the existing cytoplasmic mRNA pool and not from the newly synthesized transcripts arising after gene induction. It was also demonstrated that mechanisms of random movement were predominant in mediating the efficient translocation of mRNA in the eukaryotic cell.

The flow of genetic information in eukaryotic cells occurs through the nucleocytoplasmic translocation of mRNAs. Knowledge of in vivo messenger RNA export kinetics remains poor in comparison with that of protein transport. We have established a mammalian system that allowed the real-time visualization and quantification of large single mRNA-protein complexes (mRNPs) during export. The in vivo dynamics of bulk mRNP transport and export, from transcription to the nuclear pore complex (NPC), occurred within a 5-40 minute time frame, with no NPC pile-up. mRNP export was rapid (about 0.5 s) and kinetically faster than nucleoplasmic diffusion. Export inhibition demonstrated that mRNA-NPC interactions were independent of ongoing export. Nucleoplasmic transport dynamics of intron-containing and intronless mRNAs were similar, yet an intron did increase export efficiency. Here we provide visualization and analysis at the single mRNP level of the various steps in nuclear gene expression and the inter-chromatin tracks through which mRNPs diffuse, and demonstrate the kinetics of mRNP-NPC interactions and translocation.

The mechanism underlying the important role of protein kinase Cδ (PKCδ) in the apoptotic effect of etoposide in glioma cells is incompletely understood. Here, we examined the role of PKCδ in the activation of Erk1/2 by etoposide. We found that etoposide induced persistent activation of Erk1/2 and nuclear translocation of phospho-Erk1/2. MEK1 inhibitors decreased the apoptotic effect of etoposide, whereas inhibitors of p38 and JNK did not. The activation of Erk1/2 by etoposide was downstream of PKCδ since the phosphorylation of Erk1/2 was inhibited by a PKCδ-KD mutant and PKCδ small interfering RNA. We recently reported that phosphorylation of PKCδ on tyrosines 64 and 187 was essential for the apoptotic effect of etoposide. Using PKCδtyrosine mutants, we found that the phosphorylation of PKCδon these tyrosine residues, but not on tyrosine 155, was also essential for the activation of Erk1/2 by etoposide. In contrast, nuclear translocation of PKCδ was independent of its tyrosine phosphorylation and not necessary for the phosphorylation of Erk1/2. Etoposide induced down-regulation of kinase phosphatase-1 (MKP-1), which correlated with persistent phosphorylation of Erk1/2 and was dependent on the tyrosine phosphorylation of PKCδ. Moreover, silencing of MKP-1 increased the phosphorylation of Erk1/2 and the apoptotic effect of etoposide. Etoposide induced polyubiquitylation and degradation of MKP-1 that was dependent on PKCδ and on its tyrosine phosphorylation. These results indicate that distinct phosphorylation of PKCδon tyrosines 64 and 187 specifically activates the Erk1/2 pathway by the down-regulation of MKP-1, resulting in the persistent phosphorylation of Erk1/2 and cell apoptosis.

The transcriptional process is at the center of the gene expression pathway. In eukaryotes, the transcription of protein-coding genes into messenger RNAs is performed by RNA polymerase II. This enzyme is directed to bind at upstream gene sequences by the aid of transcription factors that assemble transcription-competent complexes. A series of biochemical and structural modifications render the polymerase transcriptionally active so that it can proceed from an initiation state into a functional elongating phase. Recent experimental efforts have attempted to visualize these processes as they take place on genes in living cells and to quantify the kinetics of in vivo transcription.

The doublecortin-like (DCX) domains serve as protein-interaction platforms. DCX tandem domains appear in the product of the X-linked doublecortin (DCX) gene, in retinitis pigmentosa-1 (RP1), as well as in other gene products. Mutations in the human DCX gene are associated with abnormal neuronal migration, epilepsy, and mental retardation; mutations in RP1 are associated with a form of inherited blindness, while DCDC2 has been associated with dyslectic reading disabilities. Motivated by the possible importance of this gene family, a thorough analysis to detect all family members in the mouse was conducted. The DCX-repeat gene superfamily is composed of eleven paralogs, and we cloned the DCX domains from nine different genes. Our study questioned which functions attributed to the DCX domain, are conserved among the different members. Our results suggest that the proteins with the DCX-domain have conserved and unique roles in microtubule regulation and signal transduction. All the tested proteins stimulated microtubule assembly in vitro. Proteins with tandem repeats stabilized the microtubule cytoskeleton in transfected cells, while those with single repeats localized to actin-rich subcellular structures, or the nucleus. All tested proteins interacted with components of the JNK/MAP-kinase pathway, while only a subset interacted with Neurabin 2, and a nonoverlapping group demonstrated actin association. The sub-specialization of some members due to confined intracellular localization, and protein interactions may explain the success of this superfamily.

180 BioTechniques Vol. 39, No. 2 (2005) Identifying the genes that are responsible for human genetic disorders with complex inheritance patterns (e.g., multigenic diseases) has proven to be more difficult than anticipated. Indeed, when the mode of inheritance is unknown, classical parametric linkage studies are not relevant. Nonparametric linkage analysis can help approximate the candidate loci, but this type of analysis often leads to the identification of large intervals (10–20 cM) that may contain hundreds of genes, thus rendering the candidate gene approach rather tedious (1). Adding an expression screening may significantly reduce the number of candidate genes. Microarray expression studies of the genes located within such genetic intervals should be performed on relevant tissues (2). The design of a custom microrarray containing probes of all the genes located in the intervals of interest is required to carry out such experiments. Designing such microarrays involves gathering much additional information concerning these genes, in particular, name, transcript accession numbers, or nucleotide sequences. Collecting these data manually is time-consuming and very error-prone. GeneRetriever is a Perl-based data mining tool developed to automate, accelerate, and secure the process of locally retrieving user-chosen comprehensive information about human genes or transcripts located between two genetic markers. As annotation strategies are specific for each database, we implemented a database parameter entry that allows collection of data from either the National Center for Biotechnology Information (NCBI; www.ncbi.nlm.nih. gov) or Ensembl (www.ensembl.org) databases (3,4). Then, several options make it possible to define which data should be included in the returned gene/transcript table. These options are clustered into three parts: (i) genespecific data; (ii) transcript-specific data; and (iii) expression analysis data. Gene-specific options include database (either NCBI or Ensembl) identifier, Hugo Gene Nomenclature Committee symbol (5), gene description, DNA strand (plus or minus), type of gene (known or predicted), cytogenetic localization, summary of functional annotations, Entrez Gene identifier (6), web address of either NCBI or Ensembl gene page, and the number of transcript variants. Additional transcript information is optionally available, such as Ensembl transcript or RefSeq accession numbers. Structural data, including transcript size, number of exons, and size of the longest exon, can also be added to the query. These data may be useful when working with predicted genes; for instance, the relevance of predicted genes composed of one single exon of

Microsatellites (MSs) are tracts of variable-length repeats of short DNA motifs that are abundant in the human genome and exhibit high rates of mutations in the form of insertions or deletions of the repeated motif (MS indels). Despite their prevalence, the contribution of somatic MS indels to cancer is largely unexplored due to difficulties in detecting them and assessing their significance. Here, we present a comprehensive analysis of MS indels across 20 tumor types. We characterize the overall MS indel landscape and detect genes with candidate driver MS indel events. We present two novel tools: MSMuTect for accurate detection of somatic MS indels and MSMutSig for identifying candidate cancer genes containing events at higher frequency than expected by chance. We observe high variability of the frequency of MS indels across tumors and demonstrate that the number and pattern of MS indels can accurately distinguish microsatellite stable (MSS) tumors from tumors with microsatellite instability (MSI). Applying MSMutSig across 6,788 tumors from 20 different tumor types identified 7 genes with significant MS indel hotspots: ACVR2A, RNF43, DOCK3, MSH3, ESRP1, PRDM2 and JAK1. In the four genes that have been previously implicated in cancer (ACVR2A, RNF43, JAK1 and MSH3), we identified previously unreported MS indels events. Three of the genes with significant loci - DOCK3, PRDM2 and ESRP1- had not been previously listed as cancer genes. MS indels in DOCK3, a negative regulator of the WNT pathway, were mutually exclusive with mutations in CTNNB1. MS indels in ESRP1, an RNA processing gene, correlated with alternative splicing of FGFR2, an event associated with the epithelial-to-mesenchymal transition. Overall, our comprehensive analysis of somatic MS indels across cancer highlights their importance, particularly in MSI tumors, significantly contributes to the ongoing global efforts to detect cancer genes, and may improve classification of patients into clinically-relevant subgroups. Citation Format: Yosef E. Maruvka, Kent W. Mouw, Rosa Karlic, Rasanna Parasuraman, Atanas Kamburov, Paz Polak, Nicholas J. Haradhvala, Julian M. Hess, Esther Rheinbay, Yehuda Brody, Lior Z. Braunstein, Alan D’Andrea, Michael S. Lawrence, Adam Bass, Andre Bernards, Franziska Michor, Gad Getz. The landscape of somatic microsatellite indels across cancer: detection and identification of driver events [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-280. doi:10.1158/1538-7445.AM2017-LB-280

RNA processing by the splicing machinery removes intronic sequences from pre-mRNA to generate mature mRNA transcripts. Many splicing events occur co-transcriptionally when the pre-mRNA is still associated with the transcription machinery. This mechanism raises questions regarding the number of spliceosomes associated with the pre-mRNA at a given time. In this protocol, we present a quantitative FISH approach that measures the ratio of intensities between two different spliceosomal components associated on a nascent mRNA, and compares to the number of introns in the mRNA, thereby calculating the number of spliceosome complexes assembled with each transcript.

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo is of importance. Pol II kinetics have been measured using biochemical or molecular methods.1-3 In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells.4 Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells.5, 6 Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo, it is possible to detect the actual transcription of mRNAs on the gene of interest.7, 8 The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed.9 To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs.5 In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.

GFP-tagged snRNP components reveal the dynamics and rate for spliceosome assembly in vivo., Precursor messenger RNA (pre-mRNA) splicing is catalyzed by the spliceosome, a large ribonucleoprotein (RNP) complex composed of five small nuclear RNP particles (snRNPs) and additional proteins. Using live cell imaging of GFP-tagged snRNP components expressed at endogenous levels, we examined how the spliceosome assembles in vivo. A comprehensive analysis of snRNP dynamics in the cell nucleus enabled us to determine snRNP diffusion throughout the nucleoplasm as well as the interaction rates of individual snRNPs with pre-mRNA. Core components of the spliceosome, U2 and U5 snRNPs, associated with pre-mRNA for 15–30 s, indicating that splicing is accomplished within this time period. Additionally, binding of U1 and U4/U6 snRNPs with pre-mRNA occurred within seconds, indicating that the interaction of individual snRNPs with pre-mRNA is distinct. These results are consistent with the predictions of the step-wise model of spliceosome assembly and provide an estimate on the rate of splicing in human cells.

Exported mRNAs are targeted for translation or can undergo degradation by several decay mechanisms. The 5′→3′ degradation machinery localizes to cytoplasmic P bodies (PBs). We followed the dynamic properties of PBs in vivo and investigated the mechanism by which PBs scan the cytoplasm. Using proteins of the decapping machinery, we asked whether PBs actively scan the cytoplasm or whether a diffusion-based mechanism is sufficient. Live-cell imaging showed that PBs were anchored mainly to microtubules. Quantitative single-particle tracking demonstrated that most PBs exhibited spatially confined motion dependent on microtubule motion, whereas stationary PB pairs were identified at the centrosome. Some PBs translocated in long-range movements on microtubules. PB mobility was compared with mitochondria, endoplasmic reticulum, peroxisomes, SMN bodies, and stress granules, and diffusion coefficients were calculated. Disruption of the microtubule network caused a significant reduction in PB mobility together with an induction of PB assembly. However, FRAP measurements showed that the dynamic flux of assembled PB components was not affected by such treatments. FRAP analysis showed that the decapping enzyme Dcp2 is a nondynamic PB core protein, whereas Dcp1 proteins continuously exchanged with the cytoplasm. This study reveals the mechanism of PB transport, and it demonstrates how PB assembly and disassembly integrate with the presence of an intact cytoskeleton.