Writing a wrong: Coupled RNA polymerase II transcription and RNA quality control

Processing and maturation of precursor RNA species is coupled to RNA polymerase II transcription. Co-transcriptional RNA processing helps to ensure efficient and proper capping, splicing, and 3' end processing of different RNA species to help ensure quality control of the transcriptome. Many improperly processed transcripts are not exported from the nucleus, are restricted to the site of transcription, and are in some cases degraded, which helps to limit any possibility of aberrant RNA causing harm to cellular health. These critical quality control pathways are regulated by the highly dynamic protein-protein interaction network at the site of transcription. Recent work has further revealed the extent to which the processes of transcription and RNA processing and quality control are integrated, and how critically their coupling relies upon the dynamic protein interactions that take place co-transcriptionally. This review focuses specifically on the intricate balance between 3' end processing and RNA decay during transcription termination. This article is categorized under: RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Processing > 3' End Processing RNA Processing > Splicing Mechanisms RNA Processing > Capping and 5' End Modifications

Dual Requirement for Yeast hnRNP Nab2p in mRNA poly(A) Tail Length Control and Nuclear Export

Recent studies of mRNA export factors have provided additional evidence for a mechanistic link between mRNA 3′‐end formation and nuclear export. Here, we identify Nab2p as a nuclear poly(A)‐binding protein required for both poly(A) tail length control and nuclear export of mRNA. Loss of NAB2 expression leads to hyperadenylation and nuclear accumulation of poly(A)+ RNA but, in contrast to mRNA export mutants, these defects can be uncoupled in a nab2 mutant strain. Previous studies have implicated the cytoplasmic poly(A) tail‐binding protein Pab1p in poly(A) tail length control during polyadenylation. Although cells are viable in the absence of NAB2 expression when PAB1 is overexpressed, Pab1p fails to resolve the nab2Δ hyperadenylation defect even when Pab1p is tagged with a nuclear localization sequence and targeted to the nucleus. These results indicate that Nab2p is essential for poly(A) tail length control in vivo, and we demonstrate that Nab2p activates polyadenylation, while inhibiting hyperadenylation, in the absence of Pab1p in vitro. We propose that Nab2p provides an important link between the termination of mRNA polyadenylation and nuclear export

Rex1p Deficiency Leads to Accumulation of Precursor Initiator tRNA\u3csup\u3eMet\u3c/sup\u3e and Polyadenylation of Substrate RNAs in \u3cem\u3eSaccharomyces cerevisiae\u3c/em\u3e

A synthetic genetic array was used to identify lethal and slow-growth phenotypes produced when a mutation in TRM6, which encodes a tRNA modification enzyme subunit, was combined with the deletion of any non-essential gene in Saccharomyces cerevisiae. We found that deletion of the REX1 gene resulted in a slow-growth phenotype in the trm6-504 strain. Previously, REX1 was shown to be involved in processing the 3′ ends of 5S rRNA and the dimeric tRNAArg-tRNAAsp. In this study, we have discovered a requirement for Rex1p in processing the 3′ end of tRNAiMet precursors and show that precursor tRNAiMet accumulates in a trm6-504 rex1Δ strain. Loss of Rex1p results in polyadenylation of its substrates, including tRNAiMet, suggesting that defects in 3′ end processing can activate the nuclear surveillance pathway. Finally, purified Rex1p displays Mg2+-dependent ribonuclease activity in vitro, and the enzyme is inactivated by mutation of two highly conserved amino acids

Yeast RNase III triggers polyadenylation-independent transcription termination

Transcription termination of messenger RNA (mRNA) is normally achieved by polyadenylation followed by Rat1p-dependent 5'-3' exoribonuleolytic degradation of the downstream transcript. Here we show that the yeast ortholog of the dsRNA-specific ribonuclease III (Rnt1p) may trigger Rat1p-dependent termination of RNA transcripts that fail to terminate near polyadenylation signals. Rnt1p cleavage sites were found downstream of several genes, and the deletion of RNT1 resulted in transcription readthrough. Inactivation of Rat1p impaired Rnt1p-dependent termination and resulted in the accumulation of 3' end cleavage products. These results support a model for transcription termination in which cotranscriptional cleavage by Rnt1p provides access for exoribonucleases in the absence of polyadenylation signals.This work was supported by a grant from the Canadian Institute of Health Research. S. A. is a Chercheur Boursier National of the Fonds de la Recherche en Santé du Québec. F.R. holds a New Investigator Award from the Canadian Institute of Health Research. P-É.J. holds a post-doctoral award from the IRCM training program in cancer research funded by the CIHR. J.-R.L is a research fellow of the Terry Fox Foundation through an award from the National Cancer Institute of Canada

Functional characterization of Ysh1p, the yeast endonuclease involved in 3" end processing and in transcription termination of RNA polymerase II transcripts

Eukaryotic RNA polymerase II (RNAP II) is involved in the synthesis of two major classes of transcripts: messenger RNAs (mRNAs) and small nuclear and small nucleolar RNAs. In order to be biologically functional, primary transcripts of RNAP II require extensive processing and modifications. Biogenesis of mature mRNAs involves capping at the 5’ end, splicing out of the introns and poly(A) tail addition at the 3’ end. Only correctly processed mRNAs can be exported to the cytoplasm where they act as templates for protein translation. Eukaryotic pre-mRNA 3’ end formation is initiated by endonucleolytic cleavage at the poly(A) site, followed by polyadenylation of the upstream cleavage product. In contrast, small nuclear RNA (snRNA) and small nucleolar RNA (snoRNA) precursors are cleaved at their 3’ ends, but in their mature form they are not polyadenylated. The seemingly simple reactions of 3’ end cleavage and polyadenylation are nevertheless performed by surprisingly complex protein machineries. In yeast, the pre-mRNA 3’ end processing apparatus consists of cleavage and polyadenylation factor (CPF), cleavage factor IA (CF IA) and cleavage factor IB (CF IB; reviewed in Zhao et al., 1999). The complexity of the 3’ end processing machinery is in part due to the necessity of precise RNA sequence recognition and also to the regulation in a wider transcriptional context. Both the exact mechanism of 3’ end processing, and many of the factors involved in these reactions exhibit a high level of similarity between metazoans and yeast. Cleavage and polyadenylation factors are co-transcriptionally recruited to the carboxy-terminal domain (CTD) of RNAP II and together with the cis-acting 3’ end processing signals are required for transcription termination on mRNA genes (reviewed in Buratowski, 2005; Proudfoot, 2004). The original aim of this thesis was the identification and characterization of the yeast endonuclease involved in pre-mRNA 3’ end processing. Whereas it has long been known that the poly(A) tails of mRNA are synthesized by poly(A) polymerase, the endonucleolytic activity involved in 3’ end cleavage remained enigmatic for many years. Therefore, in the beginning of this work we assigned putative endonucleolytic activity to the yeast CPF subunit Ysh1p/Brr5p and to its archaean homologue, M. jannaschii MJ1236, based on highly conserved metallo- β-lactamase and β-CASP domains present in these factors. Very little has been known about Ysh1/Brr5 protein and its role within the 3’ end processing machinery. We found that the conserved metallo-β-lactamase motif present in Ysh1p/Brr5p is essential for yeast viability in vivo, as any mutation within its conserved β-lactamase signature HXHXDH is detrimental to the cell. Although this fact underscored the functional importance of the metallo-β-lactamase motif in Ysh1p/Brr5p, it hampered further attempts to analyze the effects of such mutations. Moreover, biochemical assignment of a potential enzymatic activity to this factor in vitro was virtually impossible, as recombinant Ysh1p/Brr5p alone neither bound to RNA nor exhibited any nucleolytic activity. Consistently, specific cross-linking of the yeast 3’ end processing factors to the poly(A) site did not identify Ysh1p/Brr5p as the factor present at the cleavage site. Therefore, to better understand the role of Ysh1p/Brr5p in pre-mRNA 3’ end formation, we generated a series of conditional mutants of YSH1. Analysis of several temperature- and cold- sensitive ysh1 alleles revealed several important features of Ysh1p/Brr5p in different aspects of RNA processing and their coupling to RNAP II transcription termination and splicing. Firstly, we showed that Ysh1p/Brr5p is generally required for 3’ end cleavage and polyadenylation as well as for poly(A) site selection of ACT1 pre-mRNA. Interestingly, RNAP II transcription termination defects on a plasmid-borne CYC1 gene were observed in ysh1 mutant strains. Northern blot analysis of steady-state RNA extracted from ysh1-12 mutant cells detected read-through transcripts on several endogenous mRNA genes, confirming the general requirement of Ysh1p/Brr5p for transcription termination. Secondly, a significant proportion of RNAP II molecules failed to terminate transcription properly on SNR3 snoRNA gene locus in ysh1-12 mutant and extended transcripts produced from several snoRNA genes accumulated in this strain, pointing towards the involvement of Ysh1p/Brr5p in snoRNA 3’ end formation. Furthermore, we showed that Ysh1p/Brr5p is involved in the regulation of NRD1 mRNA levels. Interestingly, mutations in ysh1-12 strain resulted in splicing defects on mRNA and snoRNA genes, thus suggesting a function for Ysh1p/Brr5p in coupling of pre-mRNA 3’ end formation and splicing reactions in S. cerevisiae. In addition, we analyzed functions of Syc1p, a new yeast 3’ end processing subunit, which exhibits a high level of homology to the C-terminus of Ysh1p/Brr5p. Syc1p has possible regulatory functions in pre-mRNA 3’ end formation and possibly links the processing machinery to other nuclear events. Last not least, we carried out in vitro analyses of the recombinant M. jannaschii protein MJ1236, which is homologous to the β-lactamase and β-CASP domains of Ysh1p/Brr5p. Intriguingly, MJ1236 possesses also a KH-RNA binding domain, thus further suggesting a potential function of this factor in RNA metabolism. Heterogeneous expression and assaying of MJ1236 revealed its endonucleolytic activity on CYC1, ADH1 and GAL7 RNA substrates in vitro. This finding strongly implied the same type of hydrolyzing activity for its S. cerevisiae homologue Ysh1p/Brr5p. Only recently the pre-mRNA 3’ end endonucleolytic activity has been assigned to CPSF73, subunit of the mammalian 3’ end processing machinery, as based on its crystal structure and in vitro activity (Mandel et al., 2006). Because of its high level of homology to CPSF73, Ysh1p/Brr5p is now generally believed to be the 3’ end processing endonuclease in S. cerevisiae. This thesis is a record of a fascinating yet sometimes frustrating quest towards identification of the yeast pre-mRNA 3’ end processing endonuclease and understanding its functions in a wider transcriptional context

Highly efficient RNA-synthesizing system that uses isolated human mitochondria: new initiation events and in vivo-like processing patterns

A highly efficient RNA-synthesizing system with isolated HeLa cell mitochondria has been developed and characterized regarding its requirements and its products. In this system, transcription is initiated and the transcripts are processed in a way which closely reproduces the in vivo patterns. Total RNA labeling in isolated mitochondria proceeds at a constant rate for about 30 min at 37 degrees C; the estimated rate of synthesis is at least 10 to 15% of the in vivo rate. Polyadenylation of the mRNAs is less extensive in this system than in vivo. Furthermore, compared with the in vivo situation, rRNA synthesis in vitro is less efficient than mRNA synthesis. This is apparently due to a decreased rate of transcription initiation at the rRNA promoter and probably a tendency also for premature termination of the nascent rRNA chains. The 5'-end processing of rRNA also appears to be slowed down, and it is very sensitive to the incubation conditions, in contrast to mRNA processing. It is suggested that the lower efficiency and the lability of rRNA synthesis and processing in isolated mitochondria may be due to cessation of import from the cytoplasm of ribosomal proteins that play a crucial role in these processes. The formation of the light-strand-coded RNA 18 (7S RNA) is affected by high pH or high ATP concentration differently from the overall light-strand transcription. The dissociation of the two processes may have important implications for the mechanism of formation and the functional role of this unusual RNA species. The high efficiency, initiation capacity, and processing fidelity of the in vitro RNA-synthesizing system described here make it a valuable tool for the analysis of the role of nucleocytoplasmic-mitochondrial interactions in organelle gene expression

3′-End processing of pre-mRNA in eukaryotes

3′-Ends of almost all eukaryotic mRNAs are generated by endonucleolytic cleavage and addition of a poly(A) tail. In mammalian cells, the reaction depends on the sequence AAUAAA upstream of the cleavage site, a degenerate GU-rich sequence element downstream of the cleavage site and stimulatory sequences upstream of AAUAAA. Six factors have been identified that carry out the two reactions. With a single exception, they have been purified to homogeneity and cDNAs for 11 subunits have been cloned. Some of the cooperative RNA-protein and protein-protein interactions within the processing complex have been analyzed, but many details, including the identity of the endonuclease, remain unknown. Several examples of regulated polyadenylation are being analyzed at the molecular level. In the yeast Saccharomyces cerevisiae, sequences directing cleavage and polyadenylation are more degenerate than in metazoans, and a downstream element has not been identified. The list of processing factors may be complete now with approximately a dozen polypeptides, but their functions in the reaction are largely unknown. 3′-Processing is known to be coupled to transcription. This connection is thought to involve interactions of processing factors with the mRNA cap as well as with RNA polymerase I