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bioRxiv2017

Heterogeneity and Intrinsic Variation in Spatial Genome Organization

The genome is hierarchically organized in 3D space and its architecture is altered in differentiation, development and disease. Some of the general principles that determine global 3D genome organization have been established. However, the extent and nature of cell-to-cell and cell-intrinsic variability in genome architecture are poorly characterized. Here, we systematically probe the heterogeneity in genome organization in human fibroblasts by combining high-resolution Hi-C datasets and high-throughput genome imaging. Optical mapping of several hundred genome interaction pairs at the single cell level demonstrates low steady-state frequencies of colocalization in the population and independent behavior of individual alleles in single nuclei. Association frequencies are determined by genomic distance, higher-order chromatin architecture and chromatin environment. These observations reveal extensive variability and heterogeneity in genome organization at the level of single cells and alleles and they demonstrate the coexistence of a broad spectrum of chromatin and genome conformations in a cell population.

molecular biology↗

bioRxiv2017

Integrator Subunit 4 is a ‘Symplekin-like’ Scaffold that Associates with INTS9/11 to Form the Integrator Cleavage Module

Integrator (INT) is a transcriptional regulatory complex associated with RNA polymerase II that is required for the 3-end processing of both UsnRNAs and enhancer RNAs. Integrator subunits 9 (INTS9) and INTS11 constitute the catalytic core of INT and are paralogues of the cleavage and polyadenylation specificity factors CPSF100 and CPSF73. While CPSF73/100 are known to associate with a third protein called Symplekin, there is no paralog of Symplekin within INT raising the question of how INTS9/11 associate with the other INT subunits. Here, we have identified that INTS4 is a specific and conserved interaction partner of INTS9/11 that does not interact with either subunit individually. Although INTS4 has no significant homology with Symplekin, it possesses N-terminal HEAT repeats similar to Symplekin but also contains a {beta}-sheet rich C-terminal region, both of which are important to bind INTS9/11. We assess three functions of INT including UsnRNA 3-end processing, maintenance of Cajal body integrity, and formation of histone locus bodies to conclude that INTS4/9/11 are the most critical of the INT subunits for UsnRNA biogenesis. Altogether, these results indicate that INTS4/9/11 compose a heterotrimeric complex that likely represents the Integrator cleavage module responsible for its endonucleolytic activity.

molecular biology↗

bioRxiv2017

NmeCas9 is an intrinsically high-fidelity genome editing platform

BackgroundThe development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wild-type SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large (e.g. mammalian) genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large (~4.2 kb) open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs (e.g. from Staphylococcus aureus, Campylobacter jejuni, Geobacillus stearothermophilus and Neisseria meningitidis) are considerably smaller and therefore better suited for viral delivery.\n\nResultsHere we show that wild-type NmeCas9, when programmed with guide sequences of natural length (24 nucleotides), exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5-N4GATT-3), for NmeCas9 genome editing in human cells.\n\nConclusionsOur results show that NmeCas9 is a naturally high-fidelity genome editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering.

molecular biology↗

bioRxiv2017

COMPARISONS OF CODING AND NONCODING SEQUENCES TOINFER THE ORIGIN OF CODON USAGE BIAS

Codon usage bias is the preferential use of the subset of synonymous codons during translation. In this paper, the comparisons of normalized entropy and GC content between the sequence of coding regions of Escherichia coli k12 and noncoding regions (ncRNA, rRNA) of various organisms were done to shed light on the origin of the codon usage bias.The normalized entropy of the coding regions was found significantly higher than the noncoding regions, suggesting the role of the translation process in shaping codon usage bias. Further, when the position specific GC content of both coding and noncoding regions was analyzed, the GC2 content in coding regions was lower than GC1 and GC2 while in noncoding regions, the GC1, GC2, GC3 contents were approximately equal. This discrepancy is explained by the biased mutation coupled with the presence and absence of selection pressure. The accumulation of CG content occurs in the sequences due to mutation bias in DNA repair and recombination process. In noncoding regions, the mutation is harmful and thus, selected against while due to the degeneracy of codons in coding regions, a mutation in GC3 is neutral and hence, not selected. Thus, the accumulation of GC content occurs in coding regions, and thus codon usage bias occurs.

molecular biology↗

bioRxiv2017

DNA-PK facilitates HIV transcription by regulating the activity of RNA polymerase II and the recruitment of transcription machinery at HIV LTR

Despite the use of highly effective antiretroviral therapy (HAART), the presence of latent or transcriptionally silent proviruses prevents cure and eradication of HIV infection. These transcriptionally silent proviruses are well protected from both the immune system and HAART regimens. Thus, in order to tackle the problem of latent HIV reservoirs, it is a prerequisite to define all the pathways that regulate HIV transcription. We have previously reported that DNA-PK facilitates HIV transcription by interacting with the RNA polymerase II (RNAP II) complex recruited at HIV LTR. To extend those studies further, here we demonstrate that DNA-PK promotes HIV transcription by supporting it at several stages, including initiation, pause-release and elongation. We discovered that DNA-PK increases phosphorylation of RNAP II C-terminal domain (CTD) at serine 5 (Ser5) and serine 2 (Ser2) by both directly catalyzing and by augmenting the recruitment of P-TEFb at HIV LTR. We found that DNA-PK facilitates the establishment of euchromatin structure at HIV LTR, which further supports HIV gene expression. DNA-PK inhibition or knockdown leads to the severe impairment of HIV gene expression and conversion of euchromatin to heterochromatin at HIV LTR. It also profoundly restricts HIV replication and reactivation of latent provirus. DNA-PK promotes the recruitment of TRIM28 at LTR and facilitates the release of paused RNAP II through TRIM28 phosphorylation. The results were reproduced in cell lines belonging to both lymphoid and myeloid lineages and were confirmed in primary CD4+ T cells and peripheral blood mononuclear cells (PBMCs) from HIV-infected patients.\n\nIMPORTANCEOur results reveal the important role of DNA-PK in supporting HIV transcription, replication and latent proviral reactivation. Intriguingly, this study sheds light on an important pathway that affects HIV gene expression. These findings provide strong rationale for developing and using transcriptional inhibitors, such as DNA-PK inhibitors, as supplement to HAART regimens in order to further enhance their effectiveness and to suppress toxicity due to HIV proteins.

molecular biology↗

bioRxiv2017

CBF-1 promotes the establishment and maintenance of HIV latency by recruiting Polycomb repressive complexes, PRC1 and PRC2, at HIV LTR

The C-promoter binding factor-1 (CBF-1) is a potent and specific inhibitor of the HIV-1 LTR promoter. Here we demonstrate that the knockdown of endogenous CBF-1 in latently infected primary CD4+ T cells, using specific small hairpin RNAs (shRNA), resulted in the reactivation of latent HIV proviruses. Chromatin immunoprecipitation (ChIP) assays using latently infected primary T cells and Jurkat T-cell lines demonstrated that CBF-1 induces the establishment and maintenance of HIV latency by recruiting Polycomb Group (PcG/PRC) corepressor complexes or Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). Knockdown of CBF-1 resulted in the dissociation of PRCs corepressor complexes enhancing the recruitment of RNA polymerase II (RNAP II) at HIV LTR. Knockdown of certain components of PRC1 and PRC2 also led to the reactivation of latent proviruses. Similarly, treatment of latently infected primary CD4+ T cells with the EZH2 inhibitor, 3-deazaneplanocin A (DZNep), led to their reactivation.

molecular biology↗

bioRxiv2017

POLD replicates both strands of small kilobase-long replication bubbles initiated at a majority of human replication origins

Error-prone mutants of polymerase epsilon (POLE*) or polymerase delta (POLD1*) induce a mutator phenotype in human cancers. Here we show that the rate of mutations introduced by POLD1* is elevated by 50%, while the rate of POLE*-induced mutations is decreased twofold, within one kilobase from replication origins. These results support a model in which POLD1 replicates both the leading and the lagging strands within a kilobase from an origin. The magnitude of the mutational bias suggests that the probability of an individual origin to initiate replication exceeds 50%, which is much higher than previous estimates. Using additional data from nascent DNA sequencing and Okazaki fragments sequencing (OK-seq) experiments, we showed that a majority of origins are firing at each replication round, but the initiated replication fork does not propagate further than 1Kb in both directions. Analyses based on mutational data and on OK-seq data concordantly suggest that only approximately a quarter of fired origins result in a processive replication fork. Taken together, our results provide a new model of replication initiation.

molecular biology↗

bioRxiv2017

The role of a single non-coding nucleotide in the evolution of an epidemic African clade of Salmonella

Introductory ParagraphSalmonella enterica serovar Typhimurium ST313 is a relatively newly emerged sequence type that is causing a devastating epidemic of bloodstream infections across sub-Saharan Africa. Analysis of hundreds of Salmonella genomes has revealed that ST313 is closely-related to the ST19 group of S. Typhimurium that cause gastroenteritis across the world. The core genomes of ST313 and ST19 vary by just 1000 single-nucleotide polymorphisms (SNPs). We hypothesised that the phenotypic differences that distinguish African Salmonella from ST19 are caused by certain SNPs that directly modulate the transcription of virulence genes.\n\nHere we identified 3,597 transcriptional start sites (TSS) of the ST313 strain D23580, and searched for a gene expression signature linked to pathogenesis of Salmonella. We identified a SNP in the promoter of the pgtE gene that caused high expression of the PgtE virulence factor in African S. Typhimurium, increased the degradation of the factor B component of human complement, contributed to serum resistance and modulated virulence in the chicken infection model. The PgtE protease is known to mediate systemic infection in animal models. We propose that high levels of expression PgtE of by African S. Typhimurium ST313 promotes bacterial survival and bacterial dissemination during human infection.\n\nOur finding of a functional role for an extra-genic SNP shows that approaches used to deduce the evolution of virulence in bacterial pathogens should include a focus on non-coding regions of the genome.

molecular biology↗

bioRxiv2017

Poly(ADP-ribosyl)ation dependent changes in CTCF-chromatin binding and gene expression in breast cells

CTCF is an evolutionarily conserved and ubiquitously expressed architectural protein regulating a plethora of cellular functions via different molecular mechanisms. CTCF can undergo a number of post-translational modifications which change its properties and functions. One such modifications linked to cancer is poly(ADP-ribosyl)ation (PARylation). The highly PARylated CTCF form has an apparent molecular mass of 180 kDa (referred to as CTCF180), which can be distinguished from hypo- and non-PARylated CTCF with the apparent molecular mass of 130 kDa (referred to as CTCF130). The existing data accumulated so far have been mainly related to CTCF130. However, the properties of CTCF180 are not well understood despite its abundance in a number of primary tissues. In this study we performed ChIP-seq and RNA-seq analyses in human breast cells 226LDM, which display predominantly CTCF130 when proliferating, but CTCF180 upon cell cycle arrest. We observed that in the arrested cells the majority of sites lost CTCF, whereas fewer sites gained CTCF or remain bound (i.e. common sites). The classical CTCF binding motif was found in the lost and common, but not in the gained sites. The changes in CTCF occupancies in the lost and common sites were associated with increased chromatin densities and altered expression from the neighboring genes. Based on these results we propose a model integrating the CTCF130/180 transition with CTCF-DNA binding and gene expression changes. This study also issues an important cautionary note concerning the design and interpretation of any experiments using cells and tissues where CTCF180 may be present.

molecular biology↗

bioRxiv2017

HNRNPA1 promotes recognition of splice site decoys by U2AF2 in vivo

Alternative pre-mRNA splicing plays a major role in expanding the transcript output of human genes. This process is regulated, in part, by the interplay of trans-acting RNA binding proteins (RBPs) with myriad cis-regulatory elements scattered throughout pre-mRNAs. These molecular recognition events are critical for defining the protein coding sequences (exons) within pre-mRNAs and directing spliceosome assembly on non-coding regions (introns). One of the earliest events in this process is recognition of the 3 splice site by U2 small nuclear RNA auxiliary factor 2 (U2AF2). Splicing regulators, such as the heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), influence spliceosome assembly both in vitro and in vivo, but their mechanisms of action remain poorly described on a global scale. HNRNPA1 also promotes proof reading of 3ss sequences though a direct interaction with the U2AF heterodimer. To determine how HNRNPA1 regulates U2AF-RNA interactions in vivo, we analyzed U2AF2 RNA binding specificity using individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) in control- and HNRNPA1 over-expression cells. We observed changes in the distribution of U2AF2 crosslinking sites relative to the 3 splice sites of alternative cassette exons but not constitutive exons upon HNRNPA1 over-expression. A subset of these events shows a concomitant increase of U2AF2 crosslinking at distal intronic regions, suggesting a shift of U2AF2 to \"decoy\" binding sites. Of the many non-canonical U2AF2 binding sites, Alu-derived RNA sequences represented one of the most abundant classes of HNRNPA1-dependent decoys. Splicing reporter assays demonstrated that mutation of U2AF2 decoy sites inhibited HNRNPA1-dependent exon skipping in vivo. We propose that HNRNPA1 regulates exon definition by modulating the interaction of U2AF2 with decoy or bona fide 3 splice sites.

molecular biology↗

bioRxiv2017

SINHCAF/FAM60A links SIN3A function to the hypoxia response and its levels are predictive of cancer patient survival

The SIN3A-HDAC complex is a master transcriptional repressor, required for development but often deregulated in disease. Here, we report that the recently identified new component of this complex, SINHCAF/FAM60A, links the SIN3A-HDAC co-repressor complex function to the hypoxia response. SINHCAF Chromatin Immunoprecipitation-sequencing and gene expression analysis reveal a signature associated with the activation of the hypoxia response. We show that SINHCAF specifically repress HIF 2 mRNA and protein expression resulting in functional cellular changes in in-vitro angiogenesis, and proliferation. Analysis of patient datasets demonstrates that SINHCAF and HIF 2 mRNA levels are inversely correlated and predict contrasting outcomes for patient survival in both colon and lung cancer. This relationship is also observed in a mouse model of colon cancer, indicating an evolutionary conserved mechanism. Our analysis reveals an unexpected link between SINHCAF and cancer cell signalling via regulation of the hypoxia response that is predictive of poor patient outcome.

molecular biology↗

bioRxiv2017

N-terminal sumoylation of centromeric histone H3 variant Cse4 regulates its proteolysis to prevent mislocalization to non-centromeric chromatin

Stringent regulation of cellular levels of evolutionarily conserved centromeric histone H3 variant (CENP-A in humans, CID in flies, Cse4 in yeast) prevents its mislocalization to non-centromeric chromatin. Overexpression and mislocalization of CENP-A has been observed in cancers and leads to aneuploidy in yeast, flies, and human cells. Ubiquitin-mediated proteolysis of Cse4 by E3 ligases such as Psh1 and Sumo-Targeted Ubiquitin Ligase (STUbL) Slx5 prevent mislocalization of Cse4. Previously, we identified Siz1 and Siz2 as the major E3 ligases for sumoylation of Cse4. In this study, we identify lysine 65 (K65) in Cse4 as a SUMO site and show that sumoylation of Cse4 K65 regulates its ubiquitin-mediated proteolysis by Slx5. Strains expressing cse4 K65R exhibit reduced levels of sumoylated and ubiquitinated Cse4 in vivo. Furthermore, co-immunoprecipitation experiments reveal reduced interaction of cse4 K65R with Slx5. Defects in sumoylation of cse4 K65R contribute to increased stability and mislocalization of cse4 K65R under normal physiological conditions. Based on the increased stability of cse4 K65R in psh1{triangleup} strains but not in slx5{triangleup} strains, we conclude that Slx5 targets sumoylated Cse4 K65 for ubiquitination-mediated proteolysis independent of Psh1. In summary, we have identified and characterized the physiological role of Cse4 sumoylation and determined that sumoylation of Cse4 K65 regulates ubiquitin-mediated proteolysis and prevents mislocalization of Cse4 which is required for genome stability.

molecular biology↗

bioRxiv2017

The Mediator co-activator complex regulates Ty1 retromobility via alternative transcription start site selection

The Ty1 retrotransposons present in the genome of Saccharomyces cerevisiae belong to the large class of mobile genetic elements that replicate via an RNA intermediary and constitute a significant portion of most eukaryotic genomes. The retromobility of Ty1 is regulated by numerous host factors, including several subunits of the Mediator transcriptional co-activator complex. In spite of its known function in the nucleus, previous studies have implicated Mediator in the regulation of post-translational steps in Ty1 retromobility. To resolve this paradox, we systematically examined the effects of deleting nonessential Mediator subunits on the frequency of Ty1 retromobility and levels of retromobility intermediates. Our findings reveal that loss of distinct Mediator subunits alters Ty1 retromobility positively or negatively over a >10,000-fold range by regulating the ratio of an internal transcript, Ty1i, to the genomic Ty1 transcript. Ty1i RNA encodes a dominant negative inhibitor of Ty1 retromobility that blocks virus-like particle maturation and cDNA synthesis. These results resolve the conundrum of Mediator exerting sweeping control of Ty1 retromobility with only minor effects on the levels of Ty1 genomic RNA and the capsid protein, Gag. Since the majority of characterized intrinsic and extrinsic regulators of Ty1 retromobility alter a post-translational step(s), Mediator could play a central role in integrating signals that influence Ty1i expression to modulate retromobility.\n\nAuthor SummaryRetrotransposons are mobile genetic elements that copy their RNA genomes into DNA and insert the DNA copies into the host genome. These elements contribute to genome instability, control of host gene expression and adaptation to changing environments. Retrotransposons depend on numerous host factors for their own propagation and control. The retrovirus-like retrotransposon, Ty1, in the yeast Saccharomyces cerevisiae has been an invaluable model for retrotransposon research, and hundreds of host factors that regulate Ty1 retrotransposition have been identified. Non-essential subunits of the Mediator transcriptional co-activator complex have been identified as one set of host factors implicated in Ty1 regulation. Here, we report a systematic investigation of the effects of loss of these non-essential subunits of Mediator on Ty1 retrotransposition. Our findings reveal a heretofore unknown mechanism by which Mediator influences the balance between transcription from two promoters in Ty1 to modulate expression of an autoinhibitory transcript known as Ty1i RNA. Our results provide new insights into host control of retrotransposon activity via promoter choice and elucidate a novel mechanism by which the Mediator co-activator governs this choice.

molecular biology↗

bioRxiv2017

PTE, a novel module of mammalian Polycomb Response Elements

Polycomb Group proteins are essential epigenetic repressors. They form multiple protein complexes of which two kinds, PRC1 and PRC2, are indispensable for repression. Although much is known about their biochemical properties, how PRC1 and PRC2 are targeted to specific genes is poorly understood. Here we establish the Cyclin D2 (CCND2) oncogene as a simple model to address this question. We provide the evidence that coordinated recruitment of PRC1 and PRC2 complexes to CCND2 involves a combination of a specialized PRC1 targeting element (PTE) and an adjacent CpG-island, which together act as a human Polycomb Response Element. Chromatin immunoprecipitation analysis of CCND2 in different transcriptional states indicates that histone modifications produced by PRC1 and PRC2 are not sufficient to recruit either of the complexes. However, catalytic activity of PRC2 helps to anchor PRC1 at the PTE. Our analyses suggest that coordinated targeting of PRC1 and PRC2 complexes by juxtaposed AT-rich PTEs and CpG-islands may be a general feature of Polycomb repression in mammals.

molecular biology↗

bioRxiv2017

Transient Reduction of DNA Methylation at the Onset of Meiosis in Male Mice

The quality of germ cells depends on successful chromatin organization in meiotic prophase I (MPI). To better understand the epigenetic context of MPI we studied the dynamics of DNA methylation in wild-type male mice. We discovered an extended period of genome-wide transient reduction of DNA methylation (TRDM) during early MPI. Our data show that TRDM arises by passive demethylation in the premeiotic S phase highlighting the abundance of hemimethylated DNA in MPI. Importantly, TRDM unmasks a deficit in retrotransposon LINE-1 DNA methylation contributing to its expression in early MPI. We propose that TRDM facilitates meiosis and gamete quality control.

molecular biology↗

bioRxiv2017

Sleeping Beauty transposon integrates into non-TA dinucleotides via an alternative mechanism

Sleeping Beauty transposon (SB) is an important genetic tool for generating mutations in vertebrates. It is well known that SB exclusively integrates into TA dinucleotides. However, this \"TA law\" has never been strictly tested in large number of insertion sites after next generation sequencing was widely utilized. In this study, we analyzed 600 million pairs of Illumina sequence reads and identified 28 thousand SB insertions in non-TA sites. We recovered some non-TA sites using PCR and confirmed that at least parts of the insertions at non-TA sites are real integrations. The consensus sequence of these non-TA sites showed an asymmetric pattern distinct from the symmetric pattern of the canonical TA sites. The right side of the consensus sequence is exactly the same as the sequence of SB transposon ends, indicating interaction between the transposon DNA and the target DNA. Based on these results we suggested that SB has an alternative integration mechanism besides the canonical one to integrate its DNA into non-TA sites.\n\nHighlights[~] For the first time, we proved that Sleeping Beauty transposon can integrate into non-TA dinucleotides.\n\n [~]For the first time, we provided evidence that transposon DNA can directly interact with target DNA.\n\n [~]And for the first time, we found that a transposon can have two independent integration mechanisms.

molecular biology↗

bioRxiv2017

Thiol-linked alkylation for the metabolic sequencing of RNA

Gene expression profiling by high-throughput sequencing reveals qualitative and quantitative changes in RNA species at steady-state but obscures the intracellular dynamics of RNA transcription, processing and decay. We developed thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq), an orthogonal chemistry-based epitranscriptomics-sequencing technology that uncovers 4-thiouridine (s4U)-incorporation in RNA species at single-nucleotide resolution. In combination with well-established metabolic RNA labeling protocols and coupled to standard, low-input, high-throughput RNA sequencing methods, SLAM-seq enables rapid access to RNA polymerase II-dependent gene expression dynamics in the context of total RNA. When applied to mouse embryonic stem cells, SLAM-seq provides global and transcript-specific insights into pluripotency-associated gene expression. We validated the method by showing that the RNA-polymerase II-dependent transcriptional output scales with Oct4/Sox2/Nanog-defined enhancer activity; and we provide quantitative and mechanistic evidence for transcript-specific RNA turnover mediated by post-transcriptional gene regulatory pathways initiated by microRNAs and N6-methyladenosine. SLAM-seq facilitates the dissection of fundamental mechanisms that control gene expression in an accessible, cost-effective, and scalable manner.\n\nOne Sentence SummaryChemical nucleotide-analog derivatization provides global insights into transcriptional and post-transcriptional gene regulation

molecular biology↗

bioRxiv2017

The RS Domain of Human CFIm68 Plays a Key Role in Selection Between Alternative Sites of Pre-mRNA Cleavage and Polyadenylation

Many eukaryotic protein-coding genes give rise to alternative mRNA isoforms with identical protein-coding capacities but which differ in the extents of their 3{acute} untranslated regions (3{acute}UTRs), due to the usage of alternative sites of pre-mRNA cleavage and polyadenylation. By governing the presence of regulatory 3{acute}UTR sequences, this type of alternative polyadenylation (APA) can significantly influence the stability, localisation and translation efficiency of mRNA. Though a variety of molecular mechanisms for APA have been proposed, previous studies have identified a pivotal role for the multi-subunit cleavage factor I (CFIm) in this process in mammals. Here we show that, in line with previous reports, depletion of the CFIm 68 kDa subunit (CFIm68) by CRISPR/Cas9-mediated gene disruption in HEK293 cells leads to a shift towards the use of promoter-proximal poly(A) sites. Using these cells as the basis for a complementation assay, we show that CFIm68 lacking its arginine/serine-rich (RS) domain retains the ability to form a nuclear complex with other CFIm subunits, but selectively lacks the capacity to restore polyadenylation at promoter-distal sites. In addition, nanoparticle-mediated analysis indicates that the RS domain is extensively phosphorylated in vivo. Overall, these results suggest that the CFIm68 RS domain makes a key regulatory contribution to APA.

molecular biology↗