Search

Breeding wheat with durable resistance to the fungal pathogen Puccinia graminis f. sp. tritici (Pgt), a major threat to cereal production, is challenging due to the rapid evolution of pathogen virulence. Increased durability and broad-spectrum resistance can be achieved by introducing more than one resistance gene, but combining numerous unlinked genes by breeding is laborious. Here we generate polygenic Pgt resistance by introducing a transgene cassette of five resistance genes into bread wheat as a single locus and show that at least four of the five genes are functional. These wheat lines are resistant to aggressive and highly virulent Pgt isolates from around the world and show very high levels of resistance in the field. The simple monogenic inheritance of this multigene locus greatly simplifies its use in breeding. However, a new Pgt isolate with virulence to several genes at this locus suggests gene stacks will need strategic deployment to maintain their effectiveness. Combining fungal-resistance genes into a single cassette enables the generation of highly resistant wheat lines.

We have identified a previously unknown mechanism of reversible high-level ethambutol (EMB) resistance in Mycobacterium tuberculosis that is caused by a reversible frameshift mutation in the M. tuberculosis orn gene. A frameshift mutation in orn produces the small-colony-variant (SCV) phenotype, but this mutation does not change the MICs of any drug for wild-type M. tuberculosis However, the same orn mutation in a low-level EMB-resistant double embB-aftA mutant (MIC = 8 μg/ml) produces an SCV with an EMB MIC of 32 μg/ml. Reversible resistance is indistinguishable from a drug-persistent phenotype, because further culture of these orn-embB-aftA SCV mutants results in rapid reversion of the orn frameshifts, reestablishing the correct orn open reading frame, returning the culture to normal colony size, and reversing the EMB MIC back to that (8 μg/ml) of the parental strain. Transcriptomic analysis of orn-embB-aftA mutants compared to wild-type M. tuberculosis identified a 27-fold relative increase in the expression of embC, which is a cellular target for EMB. Expression of embC in orn-embB-aftA mutants was also increased 5-fold compared to that in the parental embB-aftA mutant, whereas large-colony orn frameshift revertants of the orn-embB-aftA mutant had levels of embC expression similar to that of the parental embB-aftA strain. Reversible frameshift mutants may contribute to a reversible form of microbiological drug resistance in human tuberculosis.

SETX (senataxin) is an RNA/DNA helicase that has been implicated in transcriptional regulation and the DNA damage response through resolution of R-loop structures. Mutations in SETX result in either of two distinct neurodegenerative disorders. SETX dominant mutations result in a juvenile form of amyotrophic lateral sclerosis (ALS) called ALS4, whereas recessive mutations are responsible for ataxia called ataxia with oculomotor apraxia type 2 (AOA2). How mutations in the same protein can lead to different phenotypes is still unclear. To elucidate AOA2 disease mechanisms, we first examined gene expression changes following SETX depletion. We observed the effects on both transcription and RNA processing, but surprisingly observed decreased R-loop accumulation in SETX-depleted cells. Importantly, we discovered a strong connection between SETX and the macroautophagy/autophagy pathway, reflecting a direct effect on transcription of autophagy genes. We show that SETX depletion inhibits the progression of autophagy, leading to an accumulation of ubiquitinated proteins, decreased ability to clear protein aggregates, as well as mitochondrial defects. Analysis of AOA2 patient fibroblasts also revealed a perturbation of the autophagy pathway. Our work has thus identified a novel function for SETX in the regulation of autophagy, whose modulation may have a therapeutic impact for AOA2. Abbreviations: 3ʹREADS: 3ʹ region extraction and deep sequencing; ACTB: actin beta; ALS4: amyotrophic lateral sclerosis type 4; AOA2: ataxia with oculomotor apraxia type 2; APA: alternative polyadenylation; AS: alternative splicing; ATG7: autophagy-related 7; ATP6V0D2: ATPase H+ transporting V0 subunit D2; BAF: bafilomycin A(1); BECN1: beclin 1; ChIP: chromatin IP; Chloro: chloroquine; CPT: camptothecin; DDR: DNA damage response; DNMT1: DNA methyltransferase 1; DRIP: DNA/RNA IP; DSBs: double strand breaks; EBs: embryoid bodies; FTD: frontotemporal dementia; GABARAP: GABA type A receptor-associated protein; GO: gene ontology; HR: homologous recombination; HTT: huntingtin; IF: immunofluorescence; IP: immunoprecipitation; iPSCs: induced pluripotent stem cells; KD: knockdown; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MN: motor neuron; MTORC1: mechanistic target of rapamycin kinase complex 1; PASS: PolyA Site Supporting; PFA: paraformaldehyde; RNAPII: RNA polymerase II; SCA: spinocerebellar ataxia; SETX: senataxin; SMA: spinal muscular atrophy; SMN1: survival of motor neuron 1, telomeric; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TSS: transcription start site; TTS: transcription termination site; ULK1: unc-51 like autophagy activating kinase 1; WB: western blot; WIPI2: WD repeat domain, phosphoinositide interacting 2; XRN2: 5ʹ-3ʹ exoribonuclease 2.

Most eukaryotic genes produce alternative polyadenylation (APA) isoforms. Here we report that, unlike previously characterized cell lineages, differentiation of syncytiotrophoblast (SCT), a cell type critical for hormone production and secretion during pregnancy, elicits widespread transcript shortening through APA in 3’UTRs and in introns. This global APA change is observed in multiple in vitro trophoblast differentiation models, and in single cells from placentas at different stages of pregnancy. Strikingly, the transcript shortening is unrelated to cell proliferation, a feature previously associated with APA control, but instead accompanies increased secretory functions. We show that 3’UTR shortening leads to transcripts with higher mRNA stability, which augments transcriptional activation, especially for genes involved in secretion. Moreover, this mechanism, named secretion-coupled APA (SCAP), is also executed in B cell differentiation to plasma cells. Together, our data indicate that SCAP tailors the transcriptome during formation of secretory cells, boosting their protein production and secretion capacity., Alternative polyadenylation generates multiple mRNA isoforms with different 3′ UTR sizes. Here the authors report global 3′ UTR shortening that is coupled to increased expression of secretory pathway genes in secretory cell differentiation.

Alternative polyadenylation (APA) is a mechanism that generates multiple mRNA isoforms with different 3′UTRs and/or coding sequences from a single gene. Here, using 3′ region extraction and deep sequencing (3′READS), we have systematically mapped cleavage and polyadenylation sites (PASs) in Drosophila melanogaster, expanding the total repertoire of PASs previously identified for the species, especially those located in A-rich genomic sequences. Cis-element analysis revealed distinct sequence motifs around fly PASs when compared to mammalian ones, including the greater enrichment of upstream UAUA elements and the less prominent presence of downstream UGUG elements. We found that over 75% of mRNA genes in Drosophila melanogaster undergo APA. The head tissue tends to use distal PASs when compared to the body, leading to preferential expression of APA isoforms with long 3′UTRs as well as with distal terminal exons. The distance between the APA sites and intron location of PAS are important parameters for APA difference between body and head, suggesting distinct PAS selection contexts. APA analysis of the RpII215C4 mutant strain, which harbors a mutant RNA polymerase II (RNAPII) with a slower elongation rate, revealed that a 50% decrease in transcriptional elongation rate leads to a mild trend of more usage of proximal, weaker PASs, both in 3′UTRs and in introns, consistent with the “first come, first served” model of APA regulation. However, this trend was not observed in the head, suggesting a different regulatory context in neuronal cells. Together, our data expand the PAS collection for Drosophila melanogaster and reveal a tissue-specific effect of APA regulation by RNAPII elongation rate.

Polyadenylation of nascent RNA by poly(A) polymerase (PAP) is important for 3′ end maturation of almost all eukaryotic mRNAs. Most mammalian genes harbor multiple polyadenylation sites (PASs), leading to expression of alternative polyadenylation (APA) isoforms with distinct functions. How poly(A) polymerases may regulate PAS usage and hence gene expression is poorly understood. Here, we show that the nuclear canonical (PAPα and PAPγ) and non-canonical (Star-PAP) PAPs play diverse roles in PAS selection and gene expression. Deficiencies in the PAPs resulted in perturbations of gene expression, with Star-PAP impacting lowly expressed mRNAs and long-noncoding RNAs to the greatest extent. Importantly, different PASs of a gene are distinctly regulated by different PAPs, leading to widespread relative expression changes of APA isoforms. The location and surrounding sequence motifs of a PAS appear to differentiate its regulation by the PAPs. We show Star-PAP-specific PAS usage regulates the expression of the eukaryotic translation initiation factor EIF4A1, the tumor suppressor gene PTEN and the long non-coding RNA NEAT1. The Star-PAP-mediated APA of PTEN is essential for DNA damage-induced increase of PTEN protein levels. Together, our results reveal a PAS-guided and PAP-mediated paradigm for gene expression in response to cellular signaling cues.

The length and complexity of tuberculosis (TB) therapy, as well as the propensity ofMycobacterium tuberculosisto develop drug resistance, are major barriers to global TB control efforts.M. tuberculosisis known to have the ability to enter into a drug-tolerant state, which may explain many of these impediments to TB treatment. We have identified a novel mechanism of genetically encoded but rapidly reversible drug-tolerance inM. tuberculosiscaused by transient frameshift mutations in a homopolymeric tract (HT) of seven cytosines (7C) in theglpKgene. Inactivating frameshift mutations associated with the 7C HT inglpKproduce small colonies that exhibit heritable multi-drug increases in minimal inhibitory concentrations and decreases in drug-dependent killing; however, reversion back to a fully drug-susceptible large-colony phenotype occurs rapidly through the introduction of additional insertions or deletions in the sameglpKHT region. These reversible frameshift mutations in the 7C HT ofM. tuberculosis glpKoccur in clinical isolates, accumulate inM. tuberculosisinfected mice with further accumulation during drug treatment, and exhibit a reversible transcriptional profile including induction ofdosRandsigHand repression ofkstRregulons, similar to that observed in otherin vitromodels ofM. tuberculosistolerance. These results suggest that GlpK phase variation may contribute to drug-tolerance, treatment failure and relapse in human TB. Drugs effective against phase-variantM. tuberculosismay hasten TB treatment and improve cure rates.SIGNIFICANCEThe ability ofM. tuberculosisto survive during prolonged treatment has been attributed to either transient stress responses or fixed heritable drug-resistance producing mutations. We show that phase-variation in theM. tuberculosis glpKgene represents a third type of resistance mechanism. The ability of theseglpKmutants to grow slowly and then rapidly revert suggests that these transiently-heritable changes may also explain how a hidden population of drug-tolerant bacteria develops during TB treatment. As a genetically trackable cause of drug-tolerance,M. tuberculosis glpKmutants provides a unique opportunity to study these phenomena at a cellular and mechanistic level. These mutants could also be used for developing drugs that target tolerant populations, leading to more rapid and effective TB treatments.

Termination of RNA polymerase II (RNAPII) transcription is associated with RNA 3′ end formation. For coding genes, termination is initiated by the cleavage/polyadenylation machinery. In contrast, a majority of noncoding transcription events in Saccharomyces cerevisiae does not rely on RNA cleavage for termination but instead terminates via a pathway that requires the Nrd1–Nab3–Sen1 (NNS) complex. Here we show that the Schizosaccharomyces pombe ortholog of Nrd1, Seb1, does not function in NNS-like termination but promotes polyadenylation site selection of coding and noncoding genes. We found that Seb1 associates with 3′ end processing factors, is enriched at the 3′ end of genes, and binds RNA motifs downstream from cleavage sites. Importantly, a deficiency in Seb1 resulted in widespread changes in 3′ untranslated region (UTR) length as a consequence of increased alternative polyadenylation. Given that Seb1 levels affected the recruitment of conserved 3′ end processing factors, our findings indicate that the conserved RNA-binding protein Seb1 cotranscriptionally controls alternative polyadenylation.

Diversification at the transcriptome 3′end is an important and evolutionarily conserved layer of gene regulation associated with differentiation and dedifferentiation processes. Here, we identify extensive transcriptome 3′end-alterations in neuroblastoma, a tumour entity with a paucity of recurrent somatic mutations and an unusually high frequency of spontaneous regression. Utilising extensive RNAi-screening we reveal the landscape and drivers of transcriptome 3′end-diversification, discovering PCF11 as critical regulator, directing alternative polyadenylation (APA) of hundreds of transcripts including a differentiation RNA-operon. PCF11 shapes inputs converging on WNT-signalling, and governs cell cycle, proliferation, apoptosis and neurodifferentiation. Postnatal PCF11 down-regulation induces a neurodifferentiation program, and low-level PCF11 in neuroblastoma associates with favourable outcome and spontaneous tumour regression. Our findings document a critical role for APA in tumorigenesis and describe a novel mechanism for cell fate reprogramming in neuroblastoma with potentially important clinical implications. We provide an interactive data repository of transcriptome-wide APA covering > 170 RNAis, and an APA-network map with regulatory hubs., In gene regulation, diversification at the transcriptome 3′end is linked to differentiation and dedifferentiation. Here, the authors discover extensive transcriptome 3′end-alterations in neuroblastoma, regulated by PCF11, and provide an interactive data repository of transcriptome-wide alternative polyadenylation.

Recent progress in the technology of transcriptome-wide high-throughput sequencing has revealed that nonsense-mediated mRNA decay (NMD) targets ~10% of physiologic transcripts for the purpose of tuning gene expression in response to various environmental conditions. Regardless of the eukaryote studied, NMD requires the ATP-dependent RNA helicase upframeshift 1 (UPF1). It was initially thought that cellular NMD targets could be defined by their binding to steady-state UPF1, which is largely hypophosphorylated. However, the propensity for steady-state UPF1 to bind RNA nonspecifically, coupled with regulated phosphorylation of UPF1 on an NMD target serving as the trigger for NMD, made it clear that it is phosphorylated UPF1 (p-UPF1), rather than steady-state UPF1, that can be used to distinguish cellular NMD targets from cellular RNAs that are not. Here, we describe the immunoprecipitation of p-UPF1 followed by RNA sequencing (p-UPF1 RIP-seq) as a transcriptome-wide approach to define physiologic NMD targets.

Alternative polyadenylation (APA) is a widespread mechanism that generates mRNA isoforms with distinct properties. Here we have systematically mapped and compared cleavage and polyadenylation sites (PASs) in two yeast species, S. cerevisiae and S. pombe. Although >80% of the mRNA genes in each species were found to display APA, S. pombe showed greater 3′ UTR size differences among APA isoforms than did S. cerevisiae. PASs in different locations of gene are surrounded with distinct sequences in both species and are often associated with motifs involved in the Nrd1-Nab3-Sen1 termination pathway. In S. pombe, strong motifs surrounding distal PASs lead to higher abundances of long 3′ UTR isoforms than short ones, a feature that is opposite in S. cerevisiae. Differences in PAS placement between convergent genes lead to starkly different antisense transcript landscapes between budding and fission yeasts. In both species, short 3′ UTR isoforms are more likely to be expressed when cells are growing in nutrient-rich media, although different gene groups are affected in each species. Significantly, 3′ UTR shortening in S. pombe coordinates with up-regulation of expression for genes involved in translation during cell proliferation. Using S. pombe strains deficient for Pcf11 or Pab2, we show that reduced expression of 3′-end processing factors lengthens 3′ UTR, with Pcf11 having a more potent effect than Pab2. Taken together, our data indicate that APA mechanisms in S. pombe and S. cerevisiae are largely different: S. pombe has many of the APA features of higher species, and Pab2 in S. pombe has a different role in APA regulation than its mammalian homolog, PABPN1.

Many long noncoding RNAs (lncRNAs) are unstable and rapidly degraded in the nucleus by the nuclear exosome. An exosome adaptor complex called NEXT (nuclear exosome targeting) functions to facilitate turnover of some of these lncRNAs. Here we show that knockdown of one NEXT subunit, Mtr4, but neither of the other two subunits, resulted in accumulation of two types of lncRNAs: prematurely terminated RNAs (ptRNAs) and upstream antisense RNAs (uaRNAs). This suggested a NEXT-independent Mtr4 function, and, consistent with this, we isolated a distinct complex containing Mtr4 and the zinc finger protein ZFC3H1. Strikingly, knockdown of either protein not only increased pt/uaRNA levels but also led to their accumulation in the cytoplasm. Furthermore, all pt/uaRNAs examined associated with active ribosomes, but, paradoxically, this correlated with a global reduction in heavy polysomes and overall repression of translation. Our findings highlight a critical role for Mtr4/ZFC3H1 in nuclear surveillance of naturally unstable lncRNAs to prevent their accumulation, transport to the cytoplasm, and resultant disruption of protein synthesis.

The heterodimeric nuclear factor (NF) 90/NF45 complex (NF90/NF45) binds nucleic acids and is a multifunctional regulator of gene expression. Here we report that depletion of NF90/NF45 restores the expression of the p53 and p21 proteins in cervical carcinoma cells infected with high-risk human papillomaviruses (HPVs). Knockdown of either NF90 or NF45 by RNA interference led to greatly elevated levels of p53 and p21 proteins in HPV-derived HeLa and SiHa cells but not in other cancerous or normal cell lines. In HeLa cells, p21 messenger-RNA (mRNA) increased concomitantly but the level of p53 mRNA was unaffected. RNA interference directed against p53 prevented the induction of both proteins. These results indicated that the upregulation of p21 is due to p53-dependent transcription, whereas p53 is regulated post-transcriptionally. Proteasome-mediated turnover of p53 is accelerated by the HPV E6 and cellular E6AP proteins. We therefore examined the hypothesis that this pathway is regulated by NF90/NF45. Indeed, depletion of NF90 attenuated the expression of E6 RNA and inhibited transcription from the HPV early promoter, revealing a new role for NF90/NF45 in HPV gene expression. The transcription inhibition was largely independent of the reduction of P-TEFb (positive transcription elongation factor b) levels caused by NF90 depletion. Consistent with p53 derepression, NF90/NF45-depleted HeLa cells displayed elevated poly ADP-ribose polymerase (PARP) cleavage and susceptibility to camptothecin-induced apoptosis. We conclude that high-risk strains of HPV utilize the cellular NF90/NF45 complex for viral E6 expression in infected cervical carcinoma cell lines. Interference with NF90/NF45 function could assist in controlling cervical carcinoma.

Alternative cleavage and polyadenylation (APA) leads to mRNA isoforms with different coding sequences (CDS) and/or 3′ untranslated regions (3′UTRs). Using 3′ Region Extraction And Deep Sequencing (3′READS), a method which addresses the internal priming and oligo(A) tail issues that commonly plague polyA site (pA) identification, we comprehensively mapped pAs in the mouse genome, thoroughly annotating 3′ ends of genes and revealing over five thousand pAs (~8% of total) flanked by A-rich sequences, which have hitherto been overlooked. About 79% of mRNA genes and 66% of long non-coding RNA (lncRNA) genes have APA; but these two gene types have distinct usage patterns for pAs in introns and upstream exons. Promoter-distal pAs become relatively more abundant during embryonic development and cell differentiation, a trend affecting pAs in both 3′-most exons and upstream regions. Upregulated isoforms generally have stronger pAs, suggesting global modulation of the 3′ end processing activity in development and differentiation.

Background Tyro3, Axl, and Mertk (TAMs) are a family of three conserved receptor tyrosine kinases that have pleiotropic roles in innate immunity and homeostasis and when overexpressed in cancer cells can drive tumorigenesis. Methods In the present study, we engineered EGFR/TAM chimeric receptors (EGFR/Tyro3, EGFR/Axl, and EGF/Mertk) with the goals to interrogate post-receptor functions of TAMs, and query whether TAMs have unique or overlapping post-receptor activation profiles. Stable expression of EGFR/TAMs in EGFR-deficient CHO cells afforded robust EGF inducible TAM receptor phosphorylation and activation of downstream signaling. Results Using a series of unbiased screening approaches, that include kinome-view analysis, phosphor-arrays, RNAseq/GSEA analysis, as well as cell biological and in vivo readouts, we provide evidence that each TAM has unique post-receptor signaling platforms and identify an intrinsic role for Axl that impinges on cell motility and invasion compared to Tyro3 and Mertk. Conclusion These studies demonstrate that TAM show unique post-receptor signatures that impinge on distinct gene expression profiles and tumorigenic outcomes. Electronic supplementary material The online version of this article (doi:10.1186/s12964-016-0142-1) contains supplementary material, which is available to authorized users.

The DNA damage response involves coordinated control of gene expression and DNA repair. Using deep sequencing, we found widespread changes of alternative cleavage and polyadenylation site usage on ultraviolet-treatment in mammalian cells. Alternative cleavage and polyadenylation regulation in the 3ʹ untranslated region is substantial, leading to both shortening and lengthening of 3ʹ untranslated regions of genes. Interestingly, a strong activation of intronic alternative cleavage and polyadenylation sites is detected, resulting in widespread expression of truncated transcripts. Intronic alternative cleavage and polyadenylation events are biased to the 5ʹ end of genes and affect gene groups with important functions in DNA damage response and cancer. Moreover, intronic alternative cleavage and polyadenylation site activation during DNA damage response correlates with a decrease in U1 snRNA levels, and is reversible by U1 snRNA overexpression. Importantly, U1 snRNA overexpression mitigates ultraviolet-induced apoptosis. Together, these data reveal a significant gene regulatory scheme in DNA damage response where U1 snRNA impacts gene expression via the U1-alternative cleavage and polyadenylation axis.

There is an error in the designations for equal authorship contributions. The first two authors, Yan Yang and Wencheng Li, should be noted as contributing equally to this work.

The RNA polymerase II largest subunit C-terminal domain consists of repeated YSPTSPS heptapeptides. The role of tyrosine-1 (Tyr1) remains incompletely understood, as, for example, mutating all Tyr1 residues to Phe (Y1F) is lethal in vertebrates but a related mutant has only a mild phenotype in S. pombe. Here we show that Y1F substitution in budding yeast resulted in a strong slow-growth phenotype. The Y1F strain was also hypersensitive to several different cellular stresses that involve MAP kinase signaling. These phenotypes were all linked to transcriptional changes, and we also identified genetic and biochemical interactions between Tyr1 and both transcription initiation and termination factors. Further studies uncovered defects related to MAP kinase I (Slt2) pathways, and we provide evidence that Slt2 phosphorylates Tyr1 in vitro and in vivo. Our study has thus identified Slt2 as a Tyr1 kinase, and in doing so provided links between stress response activation and Tyr1 phosphorylation.

Background Most mammalian genes display alternative cleavage and polyadenylation (APA). Previous studies have indicated preferential expression of APA isoforms with short 3’ untranslated regions (3’UTRs) in testes. Results By deep sequencing of the 3’ end region of poly(A) + transcripts, we report widespread shortening of 3’UTR through APA during the first wave of spermatogenesis in mouse, with 3’UTR size being the shortest in spermatids. Using genes without APA as a control, we show that shortening of 3’UTR eliminates destabilizing elements, such as U-rich elements and transposable elements, which appear highly potent during spermatogenesis. We additionally found widespread regulation of APA events in introns and exons that can affect the coding sequence of transcripts and global activation of antisense transcripts upstream of the transcription start site, suggesting modulation of splicing and initiation of transcription during spermatogenesis. Importantly, genes that display significant 3’UTR shortening tend to have functions critical for further sperm maturation, and testis-specific genes display greater 3’UTR shortening than ubiquitously expressed ones, indicating functional relevance of APA to spermatogenesis. Interestingly, genes with shortened 3’UTRs tend to have higher RNA polymerase II and H3K4me3 levels in spermatids as compared to spermatocytes, features previously known to be associated with open chromatin state. Conclusions Our data suggest that open chromatin may create a favorable cis environment for 3’ end processing, leading to global shortening of 3’UTR during spermatogenesis. mRNAs with shortened 3’UTRs are relatively stable thanks to evasion of powerful mRNA degradation mechanisms acting on 3’UTR elements. Stable mRNAs generated in spermatids may be important for protein production at later stages of sperm maturation, when transcription is globally halted. Electronic supplementary material The online version of this article (doi:10.1186/s12915-016-0229-6) contains supplementary material, which is available to authorized users.

The PAF complex (Paf1C) has been shown to regulate chromatin modifications, gene transcription, and RNA polymerase II (PolII) elongation. Here, we provide the first genome-wide profiles for the distribution of the entire complex in mammalian cells using chromatin immunoprecipitation and high throughput sequencing. We show that Paf1C is recruited not only to promoters and gene bodies, but also to regions downstream of cleavage/polyadenylation (pA) sites at 3’ ends, a profile that sharply contrasted with the yeast complex. Remarkably, we identified novel, subunit-specific links between Paf1C and regulation of alternative cleavage and polyadenylation (APA) and upstream antisense transcription using RNAi coupled with deep sequencing of the 3’ ends of transcripts. Moreover, we found that depletion of Paf1C subunits resulted in the accumulation of PolII over gene bodies, which coincided with APA. Depletion of specific Paf1C subunits led to global loss of histone H2B ubiquitylation, although there was little impact of Paf1C depletion on other histone modifications, including tri-methylation of histone H3 on lysines 4 and 36 (H3K4me3 and H3K36me3), previously associated with this complex. Our results provide surprising differences with yeast, while unifying observations that link Paf1C with PolII elongation and RNA processing, and indicate that Paf1C subunits could play roles in controlling transcript length through suppression of PolII accumulation at transcription start site (TSS)-proximal pA sites and regulating pA site choice in 3’UTRs., Author Summary Gene transcription can be regulated through multiple mechanisms, such as histone modifications that create structural changes of the chromatin leading to gene activation or suppression, or regulation of the 3’ cleavage site of the mRNA, known as alternative cleavage and polyadenylation (APA), resulting in the generation of transcript isoforms with various lengths. Here we present genome-wide subunit-specific roles of the PAF complex (Paf1C) related to both mechanisms of transcriptional regulation. Using mouse muscle cells, we show contrasting results with yeast, namely, that depletion of Paf1C subunits does not affect certain histone modifications previously associated with this complex and that the complex exhibits subunit-specific functions. We also discovered a novel role of Paf1C in APA, wherein genome-wide transcript shortening occurs after depletion of three of the subunits. However, APA varies after depletion of certain subunits, reinforcing our conclusions regarding subunit specificity. Furthermore, by comparing depletions of two subunits, we show that the accumulation of RNA polymerase II (PolII) near the transcription start site (TSS) is specifically associated with the activation of TSS-proximal pA sites observed in one depletion but not the other.

Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3’ untranslated regions (3’UTRs) and/or coding sequences. How core cleavage/polyadenylation (C/P) factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3’UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1 and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI-25/68 or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly inhibiting C/P events in introns near the 5’ end of gene and U2 suppressing those in introns with features for efficient splicing. Furthermore, PABPN1 inhibits expression of transcripts with pAs near the transcription start site (TSS), a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results support an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the TSS, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core C/P factors., Author Summary A gene can express multiple isoforms varying in the 3’ end, a phenomenon called alternative cleavage and polyadenylation, or APA. Previous studies have indicated that most eukaryotic genes display APA and the APA profile changes under different physiological and pathological conditions. However, how APA is regulated in the cell is unclear. Here using gene knockdown and high throughput sequencing we examine how APA is regulated by factors in the machinery responsible for cleavage and polyadenylation as well as factors that play essential roles in splicing. We identify several factors that play significant roles in APA in the last exon, including CFI-25/68, PABPN1, PABPC1, Fip1 and Pcf11. We also elucidate how cleavage and polyadenylation events are regulated in introns and near the transcription start site. We uncover a group of APA events that are highly regulated by core factors as well as in cell differentiation and development. We present an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the transcription start site, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core cleavage and polyadenylation factors.

Nonsense-mediated mRNA decay (NMD) couples protein synthesis to mRNA turnover. It eliminates defective transcripts and controls the abundance of certain normal mRNAs. Our study establishes a connection between NMD and the translation factor eIF5A (eukaryotic initiation factor 5A) in human cells. eIF5A modulates the synthesis of groups of proteins (the eIF5A regulon), and undergoes a distinctive two-step post-translational modification (hypusination) catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase. We show that expression of NMD-susceptible constructs was increased by depletion of the major eIF5A isoform, eIF5A1. NMD was also attenuated when hypusination was inhibited by RNA interference with either of the two eIF5A modifying enzymes, or by treatment with the drugs ciclopirox or deferiprone which inhibit deoxyhypusine hydroxylase. Transcriptome analysis by RNA-Seq identified human genes whose expression is coordinately regulated by eIF5A1, its modifying enzymes, and the pivotal NMD factor, Upf1. Transcripts encoding components of the translation system were highly represented, including some encoding ribosomal proteins controlled by alternative splicing coupled to NMD (AS-NMD). Our findings extend and strengthen the association of eIF5A with NMD, previously inferred in yeast, and show that hypusination is important for this function of human eIF5A. In addition, they advance drug-mediated NMD suppression as a therapeutic opportunity for nonsense-associated diseases. We propose that regulation of mRNA stability contributes to eIF5A's role in selective gene expression.

HIV-1 blocks apoptosis, programmed cell death, an innate defense of cells against viral invasion. However, apoptosis can be selectively reactivated in HIV-infected cells by chemical agents that interfere with HIV-1 gene expression. We studied two globally used medicines, the topical antifungal ciclopirox and the iron chelator deferiprone, for their effect on apoptosis in HIV-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates. Both medicines activated apoptosis preferentially in HIV-infected cells, suggesting that the drugs mediate escape from the viral suppression of defensive apoptosis. In infected H9 cells, ciclopirox and deferiprone enhanced mitochondrial membrane depolarization, initiating the intrinsic pathway of apoptosis to execution, as evidenced by caspase-3 activation, poly(ADP-ribose) polymerase proteolysis, DNA degradation, and apoptotic cell morphology. In isolate-infected peripheral blood mononuclear cells, ciclopirox collapsed HIV-1 production to the limit of viral protein and RNA detection. Despite prolonged monotherapy, ciclopirox did not elicit breakthrough. No viral re-emergence was observed even 12 weeks after drug cessation, suggesting elimination of the proviral reservoir. Tests in mice predictive for cytotoxicity to human epithelia did not detect tissue damage or activation of apoptosis at a ciclopirox concentration that exceeded by orders of magnitude the concentration causing death of infected cells. We infer that ciclopirox and deferiprone act via therapeutic reclamation of apoptotic proficiency (TRAP) in HIV-infected cells and trigger their preferential elimination. Perturbations in viral protein expression suggest that the antiretroviral activity of both drugs stems from their ability to inhibit hydroxylation of cellular proteins essential for apoptosis and for viral infection, exemplified by eIF5A. Our findings identify ciclopirox and deferiprone as prototypes of selectively cytocidal antivirals that eliminate viral infection by destroying infected cells. A drug-based drug discovery program, based on these compounds, is warranted to determine the potential of such agents in clinical trials of HIV-infected patients.

The human gene encoding the cleavage/polyadenylation (C/P) factor CstF-77 contains 21 exons. However, intron 3 (In3) accounts for nearly half of the gene region, and contains a C/P site (pA) with medium strength, leading to short mRNA isoforms with no apparent protein products. This intron contains a weak 5′ splice site (5′SS), opposite to the general trend for large introns in the human genome. Importantly, the intron size and strengths of 5′SS and pA are all highly conserved across vertebrates, and perturbation of these parameters drastically alters intronic C/P. We found that the usage of In3 pA is responsive to the expression level of CstF-77 as well as several other C/P factors, indicating it attenuates the expression of CstF-77 via a negative feedback mechanism. Significantly, intronic C/P of CstF-77 pre-mRNA correlates with global 3′UTR length across cells and tissues. In addition, inhibition of U1 snRNP also leads to regulation of the usage of In3 pA, suggesting that the C/P activity in the cell can be cross-regulated by splicing, leading to coordination between these two processes. Importantly, perturbation of CstF-77 expression leads to widespread alternative cleavage and polyadenylation (APA) and disturbance of cell proliferation and differentiation. Thus, the conserved intronic pA of the CstF-77 gene may function as a sensor for cellular C/P and splicing activities, controlling the homeostasis of CstF-77 and C/P activity and impacting cell proliferation and differentiation., Author Summary Autoregulation is commonly used in biological systems to control the homeostasis of certain activity, and cross-regulation coordinates multiple processes. We show that vertebrate genes encoding the cleavage/polyadenylation (C/P) factor CstF-77 contain a conserved intronic C/P site (pA) which regulates CstF-77 expression through a negative feedback loop. Since the usage of this intronic pA is also responsive to the expression of other C/P factors, the pA can function as a sensor for the cellular C/P activity. Because the CstF-77 level is important for the usage of a large number of pAs in the genome and is particularly critical for expression of genes involved in cell cycle, this autoregulatory mechanism has far-reaching implications for cell proliferation and differentiation. The human intron harboring the pA is large and has a weak 5′ splice site, both of which are also highly conserved in other vertebrates. Inhibition of U1 snRNP, which recognizes the 5′ splice site of intron, leads to upregulation of the intronic pA isoform of CstF-77 gene, suggesting that the C/P activity in the cell can be cross-regulated by splicing, leading to coordination between these two processes.

Despite the many advantages of Caenorhabditis elegans, biochemical approaches to study tissue-specific gene expression in post-embryonic stages are challenging. Here, we report a novel experimental approach for efficient determination of tissue-specific transcriptomes involving the rapid release and purification of nuclei from major tissues of post-embryonic animals by fluorescence-activated nuclei sorting (FANS), followed by deep sequencing of linearly amplified 3'-end regions of transcripts (3'-end-seq). We employed these approaches to compile the transcriptome of the developed C. elegans intestine and used this to analyse tissue-specific cleavage and polyadenylation. In agreement with intestinal-specific gene expression, highly expressed genes have enriched GATA-elements in their promoter regions and their functional properties are associated with processes that are characteristic for the intestine. We systematically mapped pre-mRNA cleavage and polyadenylation sites, or polyA sites, including more than 3000 sites that have previously not been identified. The detailed analysis of the 3'-ends of the nuclear mRNA revealed widespread alternative polyA site use (APA) in intestinally expressed genes. Importantly, we found that intestinal polyA sites that undergo APA tend to have U-rich and/or A-rich upstream auxiliary elements that may contribute to the regulation of 3'-end formation in the intestine.

Transcriptional activity regulates alternative cleavage and polyadenylation Transcriptomic and epigenomic data, as well as reporter and nuclear run-on assays collectively show that transcriptional activity regulates the relative abundance of alternative polyadenylation isoforms, indicating general coupling of 3′ end processing to transcription., Using RNA-seq and exon array data for a large number of human and mouse tissues and cells, we identified a general correlation between relative expression of alternative polyadenylation (APA) isoforms and gene expression level: short 3′UTR isoforms are relatively more abundant when genes are highly expressed whereas long 3′UTR isoforms are relatively more abundant when genes are lowly expressed. Using reporter assays with different promoters, we found that induction of transcription leads to more usage of promoter-proximal polyA sites, suggesting modulation of 3′ end processing efficiency by transcriptional activity. Global analysis and reporter-based assays further revealed that regulation of polyA site choice by transcription takes place when genes are regulated under different cell conditions. Using global and reporter-based nuclear run-on assays, we found that highly expressed genes tend to have more RNA polymerase II pausing at promoter-proximal polyA sites, as compared with lowly expressed genes, supporting the notion that the efficiency of 3′ end processing is coupled to transcriptional activity. Highly expressed genes have a lower nucleosome level but higher H3K4me3 and H3K36me3 levels at promoter-proximal polyA sites relative to distal ones, as compared with lowly expressed genes, indicating that transcriptional activity impacts 3′ end processing and regulation of APA leaves epigenetic signatures., Genes containing multiple pre-mRNA cleavage and polyadenylation sites, or polyA sites, express mRNA isoforms with variable 3′ untranslated regions (UTRs). By systematic analysis of human and mouse transcriptomes, we found that short 3′UTR isoforms are relatively more abundant when genes are highly expressed whereas long 3′UTR isoforms are relatively more abundant when genes are lowly expressed. Reporter assays indicated that polyA site choice can be modulated by transcriptional activity through the gene promoter. Using global and reporter-based nuclear run-on assays, we found that RNA polymerase II is more likely to pause at the polyA site of highly expressed genes than that of lowly expressed ones. Moreover, highly expressed genes tend to have a lower level of nucleosome but higher H3K4me3 and H3K36me3 levels at promoter-proximal polyA sites relative to distal ones. Taken together, our results indicate that polyA site usage is generally coupled to transcriptional activity, leading to regulation of alternative polyadenylation by transcription.