Nutrient-dependent control of RNA polymerase II elongation rate regulates specific gene expression programs by alternative polyadenylation.

Transcription by RNA polymerase II (RNAPII) is a dynamic process with frequent variations in the elongation rate. However, the physiological relevance of variations in RNAPII elongation kinetics has remained unclear. Here we show in yeast that a RNAPII mutant that reduces the transcription elongation rate causes widespread changes in alternative polyadenylation (APA). We unveil two mechanisms by which APA affects gene expression in the slow mutant: 3' UTR shortening and gene derepression by premature transcription termination of upstream interfering noncoding RNAs. Strikingly, the genes affected by these mechanisms are enriched for functions involved in phosphate uptake and purine synthesis, processes essential for maintenance of the intracellular nucleotide pool. As nucleotide concentration regulates transcription elongation, our findings argue that RNAPII is a sensor of nucleotide availability and that genes important for nucleotide pool maintenance have adopted regulatory mechanisms responsive to reduced rates of transcription elongation.

Repression of pervasive antisense transcription is the primary role of fission yeast RNA polymerase II CTD serine 2 phosphorylation.

The RNA polymerase II carboxy-terminal domain (CTD) consists of conserved heptapeptide repeats that can be phosphorylated to influence distinct stages of the transcription cycle, including RNA processing. Although CTD-associated proteins have been identified, phospho-dependent CTD interactions have remained elusive. Proximity-dependent biotinylation (PDB) has recently emerged as an alternative approach to identify protein-protein associations in the native cellular environment. In this study, we present a PDB-based map of the fission yeast RNAPII CTD interactome in living cells and identify phospho-dependent CTD interactions by using a mutant in which Ser2 was replaced by alanine in every repeat of the fission yeast CTD. This approach revealed that CTD Ser2 phosphorylation is critical for the association between RNAPII and the histone methyltransferase Set2 during transcription elongation, but is not required for 3' end processing and transcription termination. Accordingly, loss of CTD Ser2 phosphorylation causes a global increase in antisense transcription, correlating with elevated histone acetylation in gene bodies. Our findings reveal that the fundamental role of CTD Ser2 phosphorylation is to establish a chromatin-based repressive state that prevents cryptic intragenic transcription initiation.

PABPN1 prevents the nuclear export of an unspliced RNA with a constitutive transport element and controls human gene expression via intron retention.

Intron retention is a type of alternative splicing where one or more introns remain unspliced in a polyadenylated transcript. Although many viral systems are known to translate proteins from mRNAs with retained introns, restriction mechanisms generally prevent export and translation of incompletely spliced mRNAs. Here, we provide evidence that the human nuclear poly(A)-binding protein, PABPN1, functions in such restrictions. Using a reporter construct in which nuclear export of an incompletely spliced mRNA is enhanced by a viral constitutive transport element (CTE), we show that PABPN1 depletion results in a significant increase in export and translation from the unspliced CTE-containing transcript. Unexpectedly, we find that inactivation of poly(A)-tail exosome targeting by depletion of PAXT components had no effect on export and translation of the unspliced reporter mRNA, suggesting a mechanism largely independent of nuclear RNA decay. Interestingly, a PABPN1 mutant selectively defective in stimulating poly(A) polymerase elongation strongly enhanced the expression of the unspliced, but not of intronless, reporter transcripts. Analysis of RNA-seq data also revealed that PABPN1 controls the expression of many human genes via intron retention. Notably, PABPN1-dependent intron retention events mostly affected 3'-terminal introns and were insensitive to PAXT and NEXT deficiencies. Our findings thus disclose a role for PABPN1 in restricting nuclear export of intron-retained transcripts and reinforce the interdependence between terminal intron splicing, 3' end processing, and polyadenylation.

The Nrd1-like protein Seb1 coordinates cotranscriptional 3' end processing and polyadenylation site selection.

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.

Senataxin homologue Sen1 is required for efficient termination of RNA polymerase III transcription.

R-loop disassembly by the human helicase Senataxin contributes to genome integrity and to proper transcription termination at a subset of RNA polymerase II genes. Whether Senataxin also contributes to transcription termination at other classes of genes has remained unclear. Here, we show that Sen1, one of two fission yeast homologues of Senataxin, promotes efficient termination of RNA polymerase III (RNAP3) transcription in vivo. In the absence of Sen1, RNAP3 accumulates downstream of RNAP3-transcribed genes and produces long exosome-sensitive 3'-extended transcripts. Importantly, neither of these defects was affected by the removal of R-loops. The finding that Sen1 acts as an ancillary factor for RNAP3 transcription termination in vivo challenges the pre-existing view that RNAP3 terminates transcription autonomously. We propose that Sen1 is a cofactor for transcription termination that has been co-opted by different RNA polymerases in the course of evolution.

Co-transcriptional RNA cleavage by Drosha homolog Pac1 triggers transcription termination in fission yeast.

Transcription termination of protein-coding genes in eukaryotic cells usually relies on a tight coordination between the cleavage and polyadenylation of the pre-mRNA, and 5'-3' degradation of the downstream nascent transcript. Here we investigated the contribution of the essential fission yeast endonuclease Pac1, a homolog of human Drosha that cleaves hairpin RNA structures, in triggering polyadenylation-independent transcription termination. Using ChIP-sequencing in Pac1-deficient cells, we found that Pac1 triggers transcription termination at snRNA and snoRNA genes as well as at specific protein-coding genes. Notably, we found that Pac1-dependent premature termination occurred at two genes encoding conserved transmembrane transporters whose expression were strongly repressed by Pac1. Analysis by genome editing indicated that a stem-loop structure in the nascent transcript directs Pac1-mediated cleavage and that the regions upstream and downstream of the Pac1 cleavage site in the targeted mRNAs were stabilized by mutation of nuclear 3'-5' and 5'-3' exonucleases, respectively. Our findings unveil a premature transcription termination pathway that uncouples co-transcriptional RNA cleavage from polyadenylation, triggering rapid nuclear RNA degradation.

Fission yeast RNA-binding proteins Puf2 and Puf4 are involved in repression of ferrireductase Frp1 expression in response to iron.

This study identifies a post-transcriptional mechanism of iron uptake regulation by Puf2 and Puf4 of the Pumilio and FBF (Puf) family of RNA-binding proteins in Schizosaccharomyces pombe. Cells expressing Puf2 and Puf4 stimulate decay of the frp1+ mRNA encoding a key enzyme of the reductive iron uptake pathway. Results consistently showed that frp1+ mRNA is stabilized in puf2Δ puf4Δ mutant cells under iron-replete conditions. As a result, puf2Δ puf4Δ cells exhibit an increased sensitivity to iron accompanied by enhanced ferrireductase activity. A pool of GFP-frp1+ 3'UTR RNAs was generated using a reporter gene containing the 3' untranslated region (UTR) of frp1+ that was under the control of a regulatable promoter. Results showed that Puf2 and Puf4 accelerate the destabilization of mRNAs containing the frp1+ 3'UTR which harbors two Pumilio response elements (PREs). Binding studies revealed that the PUM-homology RNA-binding domain of Puf2 and Puf4 expressed in Escherichia coli specifically interacts with PREs in the frp1+ 3'UTR. Using RNA immunoprecipitation in combination with reverse transcription qPCR assays, results showed that Puf2 and Puf4 interact preferentially with frp1+ mRNA under basal and iron-replete conditions, thereby contributing to inhibit Frp1 production and protecting cells against toxic levels of iron.

PDCD2 functions as an evolutionarily conserved chaperone dedicated for the 40S ribosomal protein uS5 (RPS2).

PDCD2 is an evolutionarily conserved protein with previously characterized homologs in Drosophila (zfrp8) and budding yeast (Tsr4). Although mammalian PDCD2 is essential for cell proliferation and embryonic development, the function of PDCD2 that underlies its fundamental cellular role has remained unclear. Here, we used quantitative proteomics approaches to define the protein-protein interaction network of human PDCD2. Our data revealed that PDCD2 specifically interacts with the 40S ribosomal protein uS5 (RPS2) and that the PDCD2-uS5 complex is assembled co-translationally. Loss of PDCD2 expression leads to defects in the synthesis of the small ribosomal subunit that phenocopy a uS5 deficiency. Notably, we show that PDCD2 is important for the accumulation of soluble uS5 protein as well as its incorporation into 40S ribosomal subunit. Our findings support that the essential molecular function of PDCD2 is to act as a dedicated ribosomal protein chaperone that recognizes uS5 co-translationally in the cytoplasm and accompanies uS5 to ribosome assembly sites in the nucleus. As most dedicated ribosomal protein chaperones have been identified in yeast, our study reveals that similar mechanisms exist in human cells to assist ribosomal proteins coordinate their folding, nuclear import and assembly in pre-ribosomal particles.

Proximity-dependent biotinylation mediated by TurboID to identify protein-protein interaction networks in yeast.

The use of proximity-dependent biotinylation assays coupled to mass spectrometry (PDB-MS) has changed the field of protein-protein interaction studies. However, despite the recurrent and successful use of BioID-based protein-protein interactions screening in mammalian cells, the implementation of PDB-MS in yeast has not been effective. Here, we report a simple and rapid approach in yeast to effectively screen for proximal and interacting proteins in their natural cellular environment by using TurboID, a recently described version of the BirA biotin ligase. Using the protein arginine methyltransferase Rmt3 and the RNA exosome subunits, Rrp6 and Dis3, the application of PDB-MS in yeast by using TurboID was able to recover protein-protein interactions previously identified using other biochemical approaches and provided new complementary information for a given protein bait. The development of a rapid and effective PDB assay that can systematically analyze protein-protein interactions in living yeast cells opens the way for large-scale proteomics studies in this powerful model organism.

The 40S ribosomal protein uS5 (RPS2) assembles into an extraribosomal complex with human ZNF277 that competes with the PRMT3-uS5 interaction.

Ribosomal (r)-proteins are generally viewed as ubiquitous, constitutive proteins that simply function to maintain ribosome integrity. However, findings in the past decade have led to the idea that r-proteins have evolved specialized functions beyond the ribosome. For example, the 40S ribosomal protein uS5 (RPS2) is known to form an extraribosomal complex with the protein arginine methyltransferase PRMT3 that is conserved from fission yeast to humans. However, the full scope of uS5's extraribosomal functions, including whether uS5 interacts with any other proteins, is not known. In this study, we identify the conserved zinc finger protein 277 (ZNF277) as a new uS5-associated protein by using quantitative proteomics approaches in human cells. As previously shown for PRMT3, we found that ZNF277 uses a C2H2-type zinc finger domain to recognize uS5. Analysis of protein-protein interactions in living cells indicated that the ZNF277-uS5 complex is found in the cytoplasm and the nucleolus. Furthermore, we show that ZNF277 and PRMT3 compete for uS5 binding, because overexpression of PRMT3 inhibited the formation of the ZNF277-uS5 complex, whereas depletion of cellular ZNF277 resulted in increased levels of uS5-PRMT3. Notably, our results reveal that ZNF277 recognizes nascent uS5 in the course of mRNA translation, suggesting cotranslational assembly of the ZNF277-uS5 complex. Our findings thus unveil an intricate network of evolutionarily conserved protein-protein interactions involving extraribosomal uS5, suggesting a key role for uS5 beyond the ribosome.

Comparative analysis of alternative polyadenylation in S. cerevisiae and S. pombe.

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.

Long noncoding RNA repertoire and targeting by nuclear exosome, cytoplasmic exonuclease, and RNAi in fission yeast.

Long noncoding RNAs (lncRNAs), which are longer than 200 nucleotides but often unstable, contribute a substantial and diverse portion to pervasive noncoding transcriptomes. Most lncRNAs are poorly annotated and understood, although several play important roles in gene regulation and diseases. Here we systematically uncover and analyze lncRNAs in Schizosaccharomyces pombe. Based on RNA-seq data from twelve RNA-processing mutants and nine physiological conditions, we identify 5775 novel lncRNAs, nearly 4× the previously annotated lncRNAs. The expression of most lncRNAs becomes strongly induced under the genetic and physiological perturbations, most notably during late meiosis. Most lncRNAs are cryptic and suppressed by three RNA-processing pathways: the nuclear exosome, cytoplasmic exonuclease, and RNAi. Double-mutant analyses reveal substantial coordination and redundancy among these pathways. We classify lncRNAs by their dominant pathway into cryptic unstable transcripts (CUTs), Xrn1-sensitive unstable transcripts (XUTs), and Dicer-sensitive unstable transcripts (DUTs). XUTs and DUTs are enriched for antisense lncRNAs, while CUTs are often bidirectional and actively translated. The cytoplasmic exonuclease, along with RNAi, dampens the expression of thousands of lncRNAs and mRNAs that become induced during meiosis. Antisense lncRNA expression mostly negatively correlates with sense mRNA expression in the physiological, but not the genetic conditions. Intergenic and bidirectional lncRNAs emerge from nucleosome-depleted regions, upstream of positioned nucleosomes. Our results highlight both similarities and differences to lncRNA regulation in budding yeast. This broad survey of the lncRNA repertoire and characteristics in S. pombe, and the interwoven regulatory pathways that target lncRNAs, provides a rich framework for their further functional analyses.

Proteomic profiling and functional characterization of post-translational modifications of the fission yeast RNA exosome.

The RNA exosome is a conserved multi-subunit complex essential for processing and degradation of several types of RNAs. Although many of the functions of the RNA exosome are well established, whether the activity of this complex is regulated remains unclear. Here we performed a proteomic analysis of the RNA exosome complex purified from Schizosaccharomyces pombe and identified 39 post-translational modifications (PTMs), including phosphorylation, methylation, and acetylation sites. Interestingly, most of the modifications were identified in Dis3, a catalytic subunit of the RNA exosome, as well as in the exosome-associated RNA helicase, Mtr4. Functional analysis of selected PTM sites using modification-deficient and -mimetic versions of exosome subunits revealed substitutions that affected cell growth and exosome functions. Notably, our results suggest that site-specific phosphorylation in the catalytic center of Dis3 and in the helical bundle domain of Mtr4 control their activity. Our findings support a view in which post-translational modifications fine-tune exosome activity and add a layer of regulation to RNA degradation.

A Pre-mRNA degradation pathway that selectively targets intron-containing genes requires the nuclear poly(A)-binding protein.

General discard pathways eliminate unprocessed and irregular pre-mRNAs to control the quality of gene expression. In contrast to such general pre-mRNA decay, we describe here a nuclear pre-mRNA degradation pathway that controls the expression of select intron-containing genes. We show that the fission yeast nuclear poly(A)-binding protein, Pab2, and the nuclear exosome subunit, Rrp6, are the main factors involved in this polyadenylation-dependent pre-mRNA degradation pathway. Transcriptome analysis and intron swapping experiments revealed that inefficient splicing is important to dictate susceptibility to Pab2-dependent pre-mRNA decay. We also show that negative splicing regulation can promote the poor splicing efficiency required for this pre-mRNA decay pathway, and in doing so, we identified a mechanism of cross-regulation between paralogous ribosomal proteins through nuclear pre-mRNA decay. Our findings unveil a layer of regulation in the nucleus in which the turnover of specific pre-mRNAs, besides the turnover of mature mRNAs, is used to control gene expression.

Widespread exon skipping triggers degradation by nuclear RNA surveillance in fission yeast.

Exon skipping is considered a principal mechanism by which eukaryotic cells expand their transcriptome and proteome repertoires, creating different splice variants with distinct cellular functions. Here we analyze RNA-seq data from 116 transcriptomes in fission yeast (Schizosaccharomyces pombe), covering multiple physiological conditions as well as transcriptional and RNA processing mutants. We applied brute-force algorithms to detect all possible exon-skipping events, which were widespread but rare compared to normal splicing events. Exon-skipping events increased in cells deficient for the nuclear exosome or the 5'-3' exonuclease Dhp1, and also at late stages of meiotic differentiation when nuclear-exosome transcripts decreased. The pervasive exon-skipping transcripts were stochastic, did not increase in specific physiological conditions, and were mostly present at less than one copy per cell, even in the absence of nuclear RNA surveillance and during late meiosis. These exon-skipping transcripts are therefore unlikely to be functional and may reflect splicing errors that are actively removed by nuclear RNA surveillance. The average splicing rate by exon skipping was ∼ 0.24% in wild type and ∼ 1.75% in nuclear exonuclease mutants. We also detected approximately 250 circular RNAs derived from single or multiple exons. These circular RNAs were rare and stochastic, although a few became stabilized during quiescence and in splicing mutants. Using an exhaustive search algorithm, we also uncovered thousands of previously unknown splice sites, indicating pervasive splicing; yet most of these splicing variants were cryptic and increased in nuclear degradation mutants. This study highlights widespread but low frequency alternative or aberrant splicing events that are targeted by nuclear RNA surveillance.

Long-Term Prostate Specific Antigen Stability and Predictive Factors of Failure after Permanent Seed Prostate Brachytherapy.

PURPOSE: Defining biochemical failure as nadir + 2 may overestimate cure after radiotherapy. We assessed long-term prostate specific antigen stability after low dose rate prostate brachytherapy and predictors of biochemical failure when prostate specific antigen was slowly rising below the nadir + 2 ng/ml threshold. MATERIALS AND METHODS: A total of 2,339 patients with low or intermediate risk prostate cancer received 125iodine brachytherapy from 1998 to 2010 with a minimum 3-year followup. In addition, 49.7% of the patients received 6 months of androgen deprivation. Clinical, dosimetric and prostate specific antigen data were retrieved from a prospective database. Biochemical results were classified as stable or rising prostate specific antigen (0.2 ng/ml or greater and increased 0.1 ng/ml or greater during the preceding 2 years), or biochemical failure (defined as nadir + 2). Multivariate analysis was done to identify predictors of failure used to create logistic regression models. RESULTS: At a median followup of 89 months (range 37 to 199) prostate specific antigen was stable (nadir 0.03 ng/ml and at 60 months 0.04 ng/ml) in 2,004 patients (86%) and rising (nadir 0.16 ng/ml and at 60 months 0.29 ng/ml) in 145 (6%) while biochemical failure (nadir 0.51 ng/ml, p <0.001) was noted in 190 (8%). When there was no prior androgen deprivation therapy, the prostate specific antigen nadir and prostate specific antigen at 60 months were the strongest predictors of failure (OR 20.6 and 18.3, respectively, each p <0.0001). The logistic regression model had 85% sensitivity and 98% specificity, and predicted failure in 8 of 82 men (9.8%). A second model was created for the group with androgen deprivation therapy and rising prostate specific antigen using the predictive factors prostate specific antigen at 60 months (OR 53.9, p <0.0001) and T stage (OR 0.25, p = 0.0008). This model predicted biochemical failure in 30 of 56 men (54%) with 85% sensitivity and 93% specificity. The 2 predictive models yield an anticipated 90% cure rate in the entire cohort. CONCLUSIONS: Brachytherapy is highly curative with stable prostate specific antigen at a surgical ablation level in 86% of patients. Rising prostate specific antigen is rare at a 6% incidence and often innocuous.

The nuclear poly(A)-binding protein interacts with the exosome to promote synthesis of noncoding small nucleolar RNAs.

Poly(A)-binding proteins (PABPs) are important to eukaryotic gene expression. In the nucleus, the PABP PABPN1 is thought to function in polyadenylation of pre-mRNAs. Deletion of fission yeast pab2, the homolog of mammalian PABPN1, results in transcripts with markedly longer poly(A) tails, but the nature of the hyperadenylated transcripts and the mechanism that leads to RNA hyperadenylation remain unclear. Here we report that Pab2 functions in the synthesis of noncoding RNAs, contrary to the notion that PABPs function exclusively on protein-coding mRNAs. Accordingly, the absence of Pab2 leads to the accumulation of polyadenylated small nucleolar RNAs (snoRNAs). Our findings suggest that Pab2 promotes poly(A) tail trimming from pre-snoRNAs by recruiting the nuclear exosome. This work unveils a function for the nuclear PABP in snoRNA synthesis and provides insights into exosome recruitment to polyadenylated RNAs.

Polyadenylation site selection: linking transcription and RNA processing via a conserved carboxy-terminal domain (CTD)-interacting protein.

Despite the fact that the process of mRNA polyadenylation has been known for more than 40 years, a detailed understating of the mechanism underlying polyadenylation site selection is still far from complete. As 3' end processing is intimately associated with RNA polymerase II (RNAPII) transcription, factors that can successively interact with the transcription machinery and recognize cis-acting sequences on the nascent pre-mRNA would be well suited to contribute to poly(A) site selection. Studies using the fission yeast Schizosaccharomyces pombe have recently identified Seb1, a protein that shares homology with Saccharomyces cerevisiae Nrd1 and human SCAF4/8, and that is critical for poly(A) site selection. Seb1 binds to the C-terminal domain (CTD) of RNAPII via a conserved CTD-interaction domain and recognizes specific sequence motifs clustered downstream of the polyadenylation site on the uncleaved pre-mRNA. In this short review, we summarize insights into Seb1-dependent poly(A) site selection and discuss some unanswered questions regarding its molecular mechanism and conservation.

A proline-tyrosine nuclear localization signal (PY-NLS) is required for the nuclear import of fission yeast PAB2, but not of human PABPN1.

Nuclear poly(A)-binding proteins (PABPs) are evolutionarily conserved proteins that play key roles in eukaryotic gene expression. In the fission yeast Schizosaccharomyces pombe, the major nuclear PABP, Pab2, functions in the maturation of small nucleolar RNAs as well as in nuclear RNA decay. Despite knowledge about its nuclear functions, nothing is known about how Pab2 is imported into the nucleus. Here, we show that Pab2 contains a proline-tyrosine nuclear localization signal (PY-NLS) that is necessary and sufficient for its nuclear localization and function. Consistent with the role of karyopherin ß2 (Kapß2)-type receptors in the import of PY-NLS cargoes, we show that the fission yeast ortholog of human Kapß2, Kap104, binds to recombinant Pab2 and is required for Pab2 nuclear localization. The absence of arginine methylation in a basic region N-terminal to the PY-core motif of Pab2 did not affect its nuclear localization. However, in the context of a sub-optimal PY-NLS, we found that Pab2 was more efficiently targeted to the nucleus in the absence of arginine methylation, suggesting that this modification can affect the import kinetics of a PY-NLS cargo. Although a sequence resembling a PY-NLS motif can be found in the human Pab2 ortholog, PABPN1, our results indicate that neither a functional PY-NLS nor Kapß2 activity are required to promote entry of PABPN1 into the nucleus of human cells. Our findings describe the mechanism by which Pab2 is imported into the nucleus, providing the first example of a PY-NLS import system in fission yeast. In addition, this study suggests the existence of alternative or redundant nuclear import pathways for human PABPN1.

Fail-safe transcription termination: Because one is never enough.

Termination of RNA polymerase II (RNAPII) transcription is a fundamental step of gene expression that involves the release of the nascent transcript and dissociation of RNAPII from the DNA template. As transcription termination is intimately linked to RNA 3' end processing, termination pathways have a key decisive influence on the fate of the transcribed RNA. Quite remarkably, when reaching the 3' end of genes, a substantial fraction of RNAPII fail to terminate transcription, requiring the contribution of alternative or "fail-safe" mechanisms of termination to release the polymerase. This point of view covers redundant mechanisms of transcription termination and how they relate to conventional termination models. In particular, we expand on recent findings that propose a reverse torpedo model of termination, in which the 3'5' exonucleolytic activity of the RNA exosome targets transcription events associated with paused and backtracked RNAPII.