Cryptic Unstable Transcripts Research Articles

Mechanistic insights into RNA surveillance by the canonical poly(A) polymerase Pla1 of the MTREC complex

The S. pombe orthologue of the human PAXT connection, Mtl1-Red1 Core (MTREC), is an eleven-subunit complex that targets cryptic unstable transcripts (CUTs) to the nuclear RNA exosome for degradation. It encompasses the canonical poly(A) polymerase Pla1, responsible for polyadenylation of nascent RNA transcripts as part of the cleavage and polyadenylation factor (CPF/CPSF). In this study we identify and characterise the interaction between Pla1 and the MTREC complex core component Red1 and analyse the functional relevance of this interaction in vivo. Our crystal structure of the Pla1-Red1 complex shows that a 58-residue fragment in Red1 binds to the RNA recognition motif domain of Pla1 and tethers it to the MTREC complex. Structure-based Pla1-Red1 interaction mutations show that Pla1, as part of MTREC complex, hyper-adenylates CUTs for their efficient degradation. Interestingly, the Red1-Pla1 interaction is also required for the efficient assembly of the fission yeast facultative heterochromatic islands. Together, our data suggest a complex interplay between the RNA surveillance and 3’-end processing machineries.

Eisosome disruption by noncoding RNA deletion increases protein secretion in yeast.

Noncoding RNAs (ncRNAs) regulate many aspects of gene expression. We investigated how ncRNAs affected protein secretion in yeast by large-scale screening for improved endogenous invertase secretion in ncRNA deletion strains with deletion of stable unannotated transcripts (SUTs), cryptic unstable transcripts (CUTs), tRNAs, or snRNAs. We identified three candidate ncRNAs, SUT418, SUT390, and SUT125, that improved endogenous invertase secretion when deleted. As SUTs can affect expression of nearby genes, we quantified adjacent gene transcription and found that the PIL1 gene was down-regulated in the SUT125 deletion strain. Pil1 is a core component of eisosomes, nonmobile invaginations found throughout the plasma membrane. PIL1 knockout alone, or in combination with eisosome components LSP1 or SUR7, resulted in further increased secretion of invertase. Secretion of heterologous GFP was also increased upon PIL1 deletion, but this increase was signal sequence dependent. To reveal the potential for increased biopharmaceutical production, secretion of monoclonal antibody Pexelizumab scFv peptide was increased by PIL1 deletion. Global analysis of secreted proteins revealed that approximately 20% of secreted proteins, especially serine-enriched secreted proteins, including invertase, were increased upon eisosome disruption. Eisosomes are enriched with APC transporters and sphingolipids, which are essential components for secretory vesicle formation and protein sorting. Sphingolipid and serine biosynthesis pathways were up-regulated upon PIL1 deletion. We propose that increased secretion of endogenous and heterologous proteins upon PIL1 deletion resulted from sphingolipid redistribution in the plasma membrane and up-regulated sphingolipid biosynthesis. Overall, a new pathway to improve protein secretion in yeast via eisosome disruption has been identified.

The zinc-finger protein Red1 orchestrates MTREC submodules and binds the Mtl1 helicase arch domain

Cryptic unstable transcripts (CUTs) are rapidly degraded by the nuclear exosome in a process requiring the RNA helicase Mtr4 and specific adaptor complexes for RNA substrate recognition. The PAXT and MTREC complexes have recently been identified as homologous exosome adaptors in human and fission yeast, respectively. The eleven-subunit MTREC comprises the zinc-finger protein Red1 and the Mtr4 homologue Mtl1. Here, we use yeast two-hybrid and pull-down assays to derive a detailed interaction map. We show that Red1 bridges MTREC submodules and serves as the central scaffold. In the crystal structure of a minimal Mtl1/Red1 complex an unstructured region adjacent to the Red1 zinc-finger domain binds to both the Mtl1 KOW domain and stalk helices. This interaction extends the canonical interface seen in Mtr4-adaptor complexes. In vivo mutational analysis shows that this interface is essential for cell survival. Our results add to Mtr4 versatility and provide mechanistic insights into the MTREC complex.

Functional and transcriptional profiling of non-coding RNAs in yeast reveal context-dependent phenotypes and in trans effects on the protein regulatory network.

Non-coding RNAs (ncRNAs), including the more recently identified Stable Unannotated Transcripts (SUTs) and Cryptic Unstable Transcripts (CUTs), are increasingly being shown to play pivotal roles in the transcriptional and post-transcriptional regulation of genes in eukaryotes. Here, we carried out a large-scale screening of ncRNAs in Saccharomyces cerevisiae, and provide evidence for SUT and CUT function. Phenotypic data on 372 ncRNA deletion strains in 23 different growth conditions were collected, identifying ncRNAs responsible for significant cellular fitness changes. Transcriptome profiles were assembled for 18 haploid ncRNA deletion mutants and 2 essential ncRNA heterozygous deletants. Guided by the resulting RNA-seq data we analysed the genome-wide dysregulation of protein coding genes and non-coding transcripts. Novel functional ncRNAs, SUT125, SUT126, SUT035 and SUT532 that act in trans by modulating transcription factors were identified. Furthermore, we described the impact of SUTs and CUTs in modulating coding gene expression in response to different environmental conditions, regulating important biological process such as respiration (SUT125, SUT126, SUT035, SUT432), steroid biosynthesis (CUT494, SUT053, SUT468) or rRNA processing (SUT075 and snR30). Overall, these data capture and integrate the regulatory and phenotypic network of ncRNAs and protein-coding genes, providing genome-wide evidence of the impact of ncRNAs on cellular homeostasis.

The cryptic unstable transcripts are associated with developmentally regulated gene expression in blood-stage Plasmodium falciparum

ABSTRACT The tight gene expression regulation controls the development and pathogenesis of human malaria parasite Plasmodium falciparum throughout the complex life cycle. Recent studies have revealed the pervasive nascent transcripts in the genome of P. falciparum, suggesting the existence of a hidden transcriptome involved in the dynamic gene expression. However, the landscape and related biological functions of nascent non-coding RNAs (ns-ncRNAs) are still poorly explored. Here we profiled the transcription dynamics of nascent RNAs by rRNA-depleted and stranded RNA sequencing over the course of 48-h intraerythrocytic developmental cycle (IDC). We identified the genome-wide sources of a total of 2252 ns-ncRNAs, mostly originating from intergenic and untranslated regions of annotated genes. By integrating the nascent RNA abundances with ATAC-seq and ChIP-seq analysis, we uncovered the euchromatic microenvironment surrounding the ns-ncRNA loci, and revealed a positive correlation between ns-ncRNAs and corresponding mRNA abundances. Finally, by gene knock-down strategy, we showed that the cooperation of RNA exosome catalytic subunit PfDis3 and PfMtr4 cofactor played a major role in ns-ncRNAs degradation. Collectively, this study contributes to understanding of the potential roles of short-lived nascent ncRNAs in regulating gene expression in malaria parasites.

Centromeric non-coding RNA as a hidden epigenetic factor of the point centromere.

To ensure proper chromosome segregation during cell division, the centromere in many organisms is transcribed to produce a low level of long non-coding RNA to regulate the activity of the kinetochore. In the budding yeast point centromere, our recent work has shown that the level of centromeric RNAs (cenRNAs) is tightly regulated and repressed by the kinetochore protein Cbf1 and histone H2A variant H2A.ZHtz1, and de-repressed during S phase of the cell cycle. Too little or too much cenRNAs will disrupt centromere activity. Here, we discuss the current advance in the understanding of the action and regulation of cenRNAs at the point centromere of Saccharomyces cerevisiae. We further show that budding yeast cenRNAs are cryptic unstable transcripts (CUTs) that can be degraded by the nuclear RNA decay pathway. CenRNA provides an example that even CUTs, when present at the right time with the right level, can serve important cellular functions.

Disruption of the RNA exosome reveals the hidden face of the malaria parasite transcriptome

ABSTRACTAntisense transcription emerges as a key regulator of important biological processes in the human malaria parasite Plasmodium falciparum. RNA-processing factors, however, remain poorly characterized in this pathogen. Here, we purified the multiprotein RNA exosome complex of malaria parasites by affinity chromatography, using HA-tagged PfRrp4 and PfDis3 as the ligands. Seven distinct core exosome subunits (PfRrp41, PfMtr3, PfRrp42, PfRrp45, PfRrp4, PfRrp40, PfCsl4) and two exoribonuclease proteins PfRrp6 and PfDis3 are identified by mass spectrometry. Western blot analysis detects Dis3 and Rrp4 predominantly in the cytoplasmic fraction during asexual blood stage development. An inducible gene knock out of the PfDis3 subunit reveals the upregulation of structural and coding RNA, but the vast majority belongs to antisense RNA. Furthermore, we detect numerous types of cryptic unstable transcripts (CUTs) linked to virulence gene families including antisense RNA in the rif gene family. Our work highlights the limitations of steady-state RNA analysis to predict transcriptional activity and link the RNA surveillance machinery directly with post-transcriptional control and gene expression in malaria parasites.

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.

Classification of Transcription Boundary-Associated RNAs (TBARs) in Animals and Plants.

There is increasing evidence suggesting the contribution of non-coding RNAs (ncRNAs) to the phenotypic and physiological complexity of organisms. A novel ncRNA species has been identified near the transcription boundaries of protein-coding genes in eukaryotes, bacteria, and archaea. This review provides a detailed description of these transcription boundary-associated RNAs (TBARs), including their classification. Based on their genomic distribution, TBARs are divided into two major groups: promoter-associated RNAs (PARs) and terminus-associated RNAs (TARs). Depending on the sequence length, each group is further classified into long RNA species (>200 nt) and small RNA species (<200 nt). According to these rules of TBAR classification, divergent ncRNAs with confusing nomenclatures, such as promoter upstream transcripts (PROMPTs), upstream antisense RNAs (uaRNAs), stable unannotated transcripts (SUTs), cryptic unstable transcripts (CUTs), upstream non-coding transcripts (UNTs), transcription start site-associated RNAs (TSSaRNAs), transcription initiation RNAs (tiRNAs), and transcription termination site-associated RNAs (TTSaRNAs), were assigned to specific classes. Although the biogenesis pathways of PARs and TARs have not yet been clearly elucidated, previous studies indicate that some of the PARs have originated either through divergent transcription or via RNA polymerase pausing. Intriguing findings regarding the functional implications of the TBARs such as the long-range “gene looping” model, which explains their role in the transcriptional regulation of protein-coding genes, are also discussed. Altogether, this review provides a comprehensive overview of the current research status of TBARs, which will promote further investigations in this research area.

Large-scale profiling of noncoding RNA function in yeast.

Noncoding RNAs (ncRNAs) are emerging as key regulators of cellular function. We have exploited the recently developed barcoded ncRNA gene deletion strain collections in the yeast Saccharomyces cerevisiae to investigate the numerous ncRNAs in yeast with no known function. The ncRNA deletion collection contains deletions of tRNAs, snoRNAs, snRNAs, stable unannotated transcripts (SUTs), cryptic unstable transcripts (CUTs) and other annotated ncRNAs encompassing 532 different individual ncRNA deletions. We have profiled the fitness of the diploid heterozygous ncRNA deletion strain collection in six conditions using batch and continuous liquid culture, as well as the haploid ncRNA deletion strain collections arrayed individually onto solid rich media. These analyses revealed many novel environmental-specific haplo-insufficient and haplo-proficient phenotypes providing key information on the importance of each specific ncRNA in every condition. Co-fitness analysis using fitness data from the heterozygous ncRNA deletion strain collection identified two ncRNA groups required for growth during heat stress and nutrient deprivation. The extensive fitness data for each ncRNA deletion strain has been compiled into an easy to navigate database called Yeast ncRNA Analysis (YNCA). By expanding the original ncRNA deletion strain collection we identified four novel essential ncRNAs; SUT527, SUT075, SUT367 and SUT259/691. We defined the effects of each new essential ncRNA on adjacent gene expression in the heterozygote background identifying both repression and induction of nearby genes. Additionally, we discovered a function for SUT527 in the expression, 3’ end formation and localization of SEC4, an essential protein coding mRNA. Finally, using plasmid complementation we rescued the SUT075 lethal phenotype revealing that this ncRNA acts in trans. Overall, our findings provide important new insights into the function of ncRNAs.

A resource for functional profiling of noncoding RNA in the yeast Saccharomyces cerevisiae.

Eukaryotic genomes are extensively transcribed, generating many different RNAs with no known function. We have constructed 1502 molecular barcoded ncRNA gene deletion strains encompassing 443 ncRNAs in the yeast Saccharomyces cerevisiae as tools for ncRNA functional analysis. This resource includes deletions of small nuclear RNAs (snRNAs), transfer RNAs (tRNAs), small nucleolar RNAs (snoRNAs), and other annotated ncRNAs as well as the more recently identified stable unannotated transcripts (SUTs) and cryptic unstable transcripts (CUTs) whose functions are largely unknown. Specifically, deletions have been constructed for ncRNAs found in the intergenic regions, not overlapping genes or their promoters (i.e., at least 200 bp minimum distance from the closest gene start codon). The deletion strains carry molecular barcodes designed to be complementary with the protein gene deletion collection enabling parallel analysis experiments. These strains will be useful for the numerous genomic and molecular techniques that utilize deletion strains, including genome-wide phenotypic screens under different growth conditions, pooled chemogenomic screens with drugs or chemicals, synthetic genetic array analysis to uncover novel genetic interactions, and synthetic dosage lethality screens to analyze gene dosage. Overall, we created a valuable resource for the RNA community and for future ncRNA research.

Backmasking in the yeast genome: encoding overlapping information for protein-coding and RNA degradation.

Backmasking is a recording technique used to hide a sound or message in a music track in reverse, meaning that it is only audible when the record is played backwards. Analogously, the compact yeast genome encodes for diverse sources of information such as overlapping coding and non-coding transcripts, and protein-binding sites on the two complementary DNA strands. Examples are the consensus binding site sequences of the RNA-binding proteins Nrd1 and Nab3 that target non-coding transcripts for degradation. Here, by examining the overlap of stable (SUTs, stable unannotated transcripts) and unstable (CUTs, cryptic unstable transcripts) transcripts with protein-coding genes, we show that the predicted Nrd1 and Nab3-binding site sequences occur at differing frequencies. They are always depleted in the sense direction of protein-coding genes, thus avoiding degradation of the transcript. However in the antisense direction, predicted binding sites occur at high frequencies in genes with overlapping unstable ncRNAs (CUTs), so limiting the availability of non-functional transcripts. In contrast they are depleted in genes with overlapping stable ncRNAs (SUTs), presumably to avoid degrading the non-coding transcript. The protein-coding genes maintain similar amino-acid contents, but they display distinct codon usages so that Nrd1 and Nab3-binding sites can arise at differing frequencies in antisense depending on the overlapping transcript type. Our study demonstrates how yeast has evolved to encode multiple layers of information—protein-coding genes in one strand and the relative chance of degrading antisense RNA in the other strand—in the same regions of a compact genome.

The fission yeast CENP-B protein Abp1 prevents pervasive transcription of repetitive DNA elements

It is well established that eukaryotic genomes are pervasively transcribed producing cryptic unstable transcripts (CUTs). However, the mechanisms regulating pervasive transcription are not well understood. Here, we report that the fission yeast CENP-B homolog Abp1 plays an important role in preventing pervasive transcription. We show that loss of abp1 results in the accumulation of CUTs, which are targeted for degradation by the exosome pathway. These CUTs originate from different types of genomic features, but the highest increase corresponds to Tf2 retrotransposons and rDNA repeats, where they map along the entire elements. In the absence of abp1, increased RNAPII-Ser5P occupancy is observed throughout the Tf2 coding region and, unexpectedly, RNAPII-Ser5P is enriched at rDNA repeats. Loss of abp1 also results in Tf2 derepression and increased nucleolus size. Altogether these results suggest that Abp1 prevents pervasive RNAPII transcription of repetitive DNA elements (i.e., Tf2 and rDNA repeats) from internal cryptic sites.

Protein Abundance Control by Non-coding Antisense Transcription.

SummaryStable unannotated transcripts (SUTs), some of which overlap protein-coding genes in antisense direction, are a class of non-coding RNAs. While case studies have reported important regulatory roles for several of such RNAs, their general impact on protein abundance regulation of the overlapping gene is not known. To test this, we employed seamless gene manipulation to repress antisense SUTs of 162 yeast genes by using a unidirectional transcriptional terminator and a GFP tag. We found that the mere presence of antisense SUTs was not sufficient to influence protein abundance, that observed effects of antisense SUTs correlated with sense transcript start site overlap, and that the effects were generally weak and led to reduced protein levels. Antisense regulated genes showed increased H3K4 di- and trimethylation and had slightly lower than expected noise levels. Our results suggest that the functionality of antisense RNAs has gene and condition-specific components.

Survey of cryptic unstable transcripts in yeast.

BackgroundCryptic unstable transcripts (CUTs) are a largely unexplored class of nuclear exosome degraded, non-coding RNAs in budding yeast. It is highly debated whether CUT transcription has a functional role in the cell or whether CUTs represent noise in the yeast transcriptome.We sought to ascertain the extent of conserved CUT expression across a variety of Saccharomyces yeast strains to further understand and characterize the nature of CUT expression.ResultsWe sequenced the WT and rrp6Δ transcriptomes of three S.cerevisiae strains: S288c, Σ1278b, JAY291 and the S.paradoxus strain N17 and utilized a hidden Markov model to annotate CUTs in these four strains. Utilizing a four-way genomic alignment we identified a large population of CUTs with conserved syntenic expression across all four strains. By identifying configurations of gene-CUT pairs, where CUT expression originates from the gene 5’ or 3′ nucleosome free region, we observed distinct gene expression trends specific to these configurations which were most prevalent in the presence of conserved CUT expression. Divergent pairs correlate with higher expression of genes, and convergent pairs correlate with reduced gene expression.ConclusionsOur RNA-seq based method has greatly expanded upon previous CUT annotations in S.cerevisiae underscoring the extensive and pervasive nature of unstable transcription. Furthermore we provide the first assessment of conserved CUT expression in yeast and globally demonstrate possible modes of CUT-based regulation of gene expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2622-5) contains supplementary material, which is available to authorized users.

Expression of Subtelomeric lncRNAs Links Telomeres Dynamics to RNA Decay in S. cerevisiae.

Long non-coding RNAs (lncRNAs) have been shown to regulate gene expression, chromatin domains and chromosome stability in eukaryotic cells. Recent observations have reported the existence of telomeric repeats containing long ncRNAs – TERRA in mammalian and yeast cells. However, their functions remain poorly characterized. Here, we report the existence in S. cerevisiae of several lncRNAs within Y′ subtelomeric regions. We have called them subTERRA. These belong to Cryptic Unstable Transcripts (CUTs) and Xrn1p-sensitive Unstable Transcripts (XUTs) family. subTERRA transcription, carried out mainly by RNAPII, is initiated within the subtelomeric Y’ element and occurs in both directions, towards telomeres as well as centromeres. We show that subTERRA are distinct from TERRA and are mainly degraded by the general cytoplasmic and nuclear 5′- and 3′- RNA decay pathways in a transcription-dependent manner. subTERRA accumulates preferentially during the G1/S transition and in C-terminal rap1 mutant but independently of Rap1p function in silencing. The accumulation of subTERRA in RNA decay mutants coincides with telomere misregulation: shortening of telomeres, loss of telomeric clustering in mitotic cells and changes in silencing of subtelomeric regions. Our data suggest that subtelomeric RNAs expression links telomere maintenance to RNA degradation pathways.

The exosome component Rrp6 is required for RNA polymerase II termination at specific targets of the Nrd1-Nab3 pathway.

The exosome and its nuclear specific subunit Rrp6 form a 3’-5’ exonuclease complex that regulates diverse aspects of RNA biology including 3’ end processing and degradation of a variety of noncoding RNAs (ncRNAs) and unstable transcripts. Known targets of the nuclear exosome include short (<1000 bp) RNAPII transcripts such as small noncoding RNAs (snRNAs), cryptic unstable transcripts (CUTs), and some stable unannotated transcripts (SUTs) that are terminated by an Nrd1, Nab3, and Sen1 (NNS) dependent mechanism. NNS-dependent termination is coupled to RNA 3’ end processing and/or degradation by the Rrp6/exosome in yeast. Recent work suggests Nrd1 is necessary for transcriptome surveillance, regulating promoter directionality and suppressing antisense transcription independently of, or prior to, Rrp6 activity. It remains unclear whether Rrp6 is directly involved in termination; however, Rrp6 has been implicated in the 3’ end processing and degradation of ncRNA transcripts including CUTs. To determine the role of Rrp6 in NNS termination globally, we performed RNA sequencing (RNA-Seq) on total RNA and perform ChIP-exo analysis of RNA Polymerase II (RNAPII) localization. Deletion of RRP6 promotes hyper-elongation of multiple NNS-dependent transcripts resulting from both improperly processed 3’ RNA ends and faulty transcript termination at specific target genes. The defects in RNAPII termination cause transcriptome-wide changes in mRNA expression through transcription interference and/or antisense repression, similar to previously reported effects of depleting Nrd1 from the nucleus. Elongated transcripts were identified within all classes of known NNS targets with the largest changes in transcription termination occurring at CUTs. Interestingly, the extended transcripts that we have detected in our studies show remarkable similarity to Nrd1-unterminated transcripts at many locations, suggesting that Rrp6 acts with the NNS complex globally to promote transcription termination in addition to 3’ end RNA processing and/or degradation at specific targets.

The nuclear exosome is active and important during budding yeast meiosis.

Nuclear RNA degradation pathways are highly conserved across eukaryotes and play important roles in RNA quality control. Key substrates for exosomal degradation include aberrant functional RNAs and cryptic unstable transcripts (CUTs). It has recently been reported that the nuclear exosome is inactivated during meiosis in budding yeast through degradation of the subunit Rrp6, leading to the stabilisation of a subset of meiotic unannotated transcripts (MUTs) of unknown function. We have analysed the activity of the nuclear exosome during meiosis by deletion of TRF4, which encodes a key component of the exosome targeting complex TRAMP. We find that TRAMP mutants produce high levels of CUTs during meiosis that are undetectable in wild-type cells, showing that the nuclear exosome remains functional for CUT degradation, and we further report that the meiotic exosome complex contains Rrp6. Indeed Rrp6 over-expression is insufficient to suppress MUT transcripts, showing that the reduced amount of Rrp6 in meiotic cells does not directly cause MUT accumulation. Lack of TRAMP activity stabilises ∼1600 CUTs in meiotic cells, which occupy 40% of the binding capacity of the nuclear cap binding complex (CBC). CBC mutants display defects in the formation of meiotic double strand breaks (DSBs), and we see similar defects in TRAMP mutants, suggesting that a key function of the nuclear exosome is to prevent saturation of the CBC complex by CUTs. Together, our results show that the nuclear exosome remains active in meiosis and has an important role in facilitating meiotic recombination.

The Role of Ctk1 Kinase in Termination of Small Non-Coding RNAs

Transcription termination in Saccharomyces cerevisiae can be performed by at least two distinct pathways and is influenced by the phosphorylation status of the carboxy-terminal domain (CTD) of RNA polymerase II (Pol II). Late termination of mRNAs is performed by the CPF/CF complex, the recruitment of which is dependent on CTD-Ser2 phosphorylation (Ser2P). Early termination of shorter cryptic unstable transcripts (CUTs) and small nucleolar/nuclear RNAs (sno/snRNAs) is performed by the Nrd1-Nab3-Sen1 (NNS) complex that binds phosphorylated CTD-Ser5 (Ser5P) via the CTD-interacting domain (CID) of Nrd1p. In this study, mutants of the different termination pathways were compared by genome-wide expression analysis. Surprisingly, the expression changes observed upon loss of the CTD-Ser2 kinase Ctk1p are more similar to those derived from alterations in the Ser5P-dependent NNS pathway, than from loss of CTD-Ser2P binding factors. Tiling array analysis of ctk1Δ cells reveals readthrough at snoRNAs, at many cryptic unstable transcripts (CUTs) and stable uncharacterized transcripts (SUTs), but only at some mRNAs. Despite the suggested predominant role in termination of mRNAs, we observed that a CTK1 deletion or a Pol II CTD mutant lacking all Ser2 positions does not result in a global mRNA termination defect. Rather, termination defects in these strains are widely observed at NNS-dependent genes. These results indicate that Ctk1p and Ser2 CTD phosphorylation have a wide impact in termination of small non-coding RNAs but only affect a subset of mRNA coding genes.

Rrp47 functions in RNA surveillance and stable RNA processing when divorced from the exoribonuclease and exosome-binding domains of Rrp6

The eukaryotic exosome exoribonuclease Rrp6 forms a complex with Rrp47 that functions in nuclear RNA quality control mechanisms, the degradation of cryptic unstable transcripts (CUTs), and in the 3' end maturation of stable RNAs. Stable expression of Rrp47 is dependent upon its interaction with the N-terminal domain of Rrp6 (Rrp6NT). To address the function of Rrp47 independently of Rrp6, we developed a DECOID (decreased expression of complexes by overexpression of interacting domains) strategy to resolve the Rrp6/Rrp47 complex in vivo and employed mpp6Δ and rex1Δ mutants that are synthetic lethal with loss-of-function rrp47 mutants. Strikingly, Rrp47 was able to function in mpp6Δ and rex1Δ mutants when separated from the catalytic and exosome-binding domains of Rrp6, whereas a truncated Rrp47 protein lacking its C-terminal region caused a block in cell growth. Northern analyses of the conditional mutants revealed a specific block in the 3' maturation of box C/D snoRNAs in the rex1 rrp47 mutant and widespread inhibition of Rrp6-mediated RNA surveillance processes in the mpp6 rrp47 mutant. In contrast, growth analyses and RNA northern blot hybridization analyses showed no effect on the rrp47Δ mutant upon overexpression of the Rrp6NT domain. These findings demonstrate that Rrp47 and Rrp6 have resolvable functions in Rrp6-mediated RNA surveillance and processing pathways. In addition, this study reveals a redundant requirement for Rrp6 or Rex1 in snoRNA maturation and demonstrates the effective use of the DECOID strategy for the resolution and functional analysis of protein complexes.