Role of poly(A) polymerase in the cleavage and polyadenylation of mRNA precursor.

To determine the role of poly(A) polymerase in 3'-end processing of mRNA, the effect of purified poly(A) polymerase antibodies on endonucleolytic cleavage and polyadenylation was studied in HeLa nuclear extracts, using adenovirus L3 pre-mRNA as the substrate. Both Mg2+- and Mn2+-dependent reactions catalyzing addition of 200 to 250 and 400 to 800 adenylic acid residues, respectively, were inhibited by the antibodies, which suggested that the two reactions were catalyzed by the same enzyme. Anti-poly(A) polymerase antibodies also inhibited the cleavage reaction when the reaction was coupled or chemically uncoupled with polyadenylation. These antibodies also prevented formation of specific complexes between the RNA substrate and components of nuclear extracts during cleavage or polyadenylation, with the concurrent appearance of another, antibody-specific complex. These studies demonstrate that (i) previously characterized poly(A) polymerase is the enzyme responsible for addition of the poly(A) tract at the correct cleavage site and probably for the elongation of poly(A) chains and (ii) the coupling of these two 3'-end processing reactions appears to result from the potential requirement of poly(A) polymerase for the cleavage reaction. The results suggest that the specific endonuclease is associated with poly(A) polymerase in a functional complex.     

The Ref/Aly proteins are dispensable for mRNA export and development in Caenorhabditis elegans

The mRNA export pathway is highly conserved throughout evolution. We have used RNA interference (RNAi) to functionally characterize bona fide RNA export factors and components of the exon-exon junction complex (EJC) in Caenorhabditis elegans. RNAi of CeNXT1/p15, the binding partner of CeNXF1/TAP, caused early embryonic lethality, demonstrating an essential function of this gene during C. elegans development. Moreover, depletion of this protein resulted in nuclear accumulation of poly(A)(+) RNAs, supporting a direct role of NXT1/p15 in mRNA export in C. elegans. Previously, we have shown that RNAi of CeSRm160, a protein of the EJC complex, resulted in wild-type phenotype; in the present study, we demonstrate that RNAi of CeY14, another component of this complex, results in embryonic lethality. In contrast, depletion of the EJC component CeRNPS1 results in no discernible phenotype. Proteins of the REF/Aly family act as adaptor proteins mediating the recruitment of the mRNA export factor, NXF1/TAP, to mRNAs. The C. elegans genome encodes three members of the REF/Aly family. RNAi of individual Ref genes, or codepletion of two Ref genes in different combinations, resulted in wild-type phenotype. Simultaneous suppression of all three Ref genes did not compromise viability or progression through developmental stages in the affected progeny, and only caused a minor defect in larval mobility. Furthermore, no defects in mRNA export were observed upon simultaneous depletion of all three REF proteins. These results suggest the existence of multiple adaptor proteins that mediate mRNA export in C. elegans.     

Mpe1, a Zinc Knuckle Protein, Is an Essential Component of Yeast Cleavage and Polyadenylation Factor Required for the Cleavage and Polyadenylation of mRNA

In Saccharomyces cerevisiae, in vitro mRNA cleavage and polyadenylation require the poly(A) binding protein, Pab1p, and two multiprotein complexes: CFI (cleavage factor I) and CPF (cleavage and polyadenylation factor). We characterized a novel essential gene, MPE1 (YKL059c), which interacts genetically with the PCF11 gene encoding a subunit of CFI. Mpe1p is an evolutionarily conserved protein, a homolog of which is encoded by the human genome. The protein sequence contains a putative RNA-binding zinc knuckle motif. MPE1 is implicated in the choice of ACT1 mRNA polyadenylation site in vivo. Extracts from a conditional mutant, mpe1-1, or from a wild-type extract immunoneutralized for Mpe1p are defective in 3'-end processing. We used the tandem affinity purification (TAP) method on strains TAP-tagged for Mpe1p or Pfs2p to show that Mpe1p, like Pfs2p, is an integral subunit of CPF. Nevertheless a stable CPF, devoid of Mpe1p, was purified from the mpe1-1 mutant strain, showing that Mpe1p is not directly involved in the stability of this complex. Consistently, Mpe1p is also not necessary for the processive polyadenylation, nonspecific for the genuine pre-mRNA 3' end, displayed by the CPF alone. However, a reconstituted assay with purified CFI, CPF, and the recombinant Pab1p showed that Mpe1p is strictly required for the specific cleavage and polyadenylation of pre-mRNA. These results show that Mpe1p plays a crucial role in 3' end formation probably by promoting the specific link between the CFI/CPF complex and pre-mRNA.     

Model for tissue specific Calcitonin/CGRP-I RNA processing from in vitro experiments.

The Calcitonin/CGRP-I (CALC-I) gene is known to be expressed in a tissue specific fashion resulting in the production of Calcitonin mRNA in thyroid C-cells and CGRP-I mRNA in particular nerve cells. The alternative RNA processing reactions include splicing of exons 1, 2 and 3 to exon 4 and poly (A) addition at exon 4 (Calcitonin mRNA) or splicing of exons 1, 2 and 3 to exons 5 and 6 and poly (A) addition at exon 6 (CGRP-I mRNA). Using a model precursor RNA containing the exon 3 to exon 5 region of the human CALC-I gene we have investigated the Calcitonin- and CGRP-I mRNA-specific processing reactions in vitro, in nuclear extracts of Hela, PC12 and Ewing-1B cells, respectively. Extracts of PC12- and Ewing-1B cells were expected to perform CGRP mRNA-specific splicing, whereas Calcitonin mRNA specific processing was expected to occur in Hela cell extracts. Surprisingly, CGRP mRNA-specific splicing of exon 3 to exon 5 was the predominant reaction in all three extracts. Significant Calcitonin mRNA-specific splicing of exon 3 to exon 4 only took place upon elimination of the dominant downstream 3' splice site used in CGRP mRNA-specific splicing. This elimination occurs most definitively by cleavage at the Calcitonin mRNA specific poly (A) site at exon 4 which may then be the major regulatory mechanism for tissue-specific expression of the CALC-I gene.     

Synthetic lethal interactions with conditional poly(A) polymerase alleles identify LCP5, a gene involved in 18S rRNA maturation.

To identify new genes involved in 3'-end formation of mRNAs in Saccharomyces cerevisiae, we carried out a screen for synthetic lethal mutants with the conditional poly(A) polymerase allele, pap1-7. Five independent temperature-sensitive mutations called Icp1 to Icp5 (for lethal with conditional pap1 allele) were isolated. Here, we describe the characterization of the essential gene LCP5 which codes for a protein with a calculated molecular mass of 40.8 kD. Unexpectedly, we found that mutations in LCP5 caused defects in pre-ribosomal RNA (pre-rRNA) processing, whereas mRNA 3'-end formation in vitro was comparable to wild-type. Early cleavage steps (denoted A0 to A2) that lead to the production of mature 18S rRNA were impaired. In vivo depletion of Lcp5p also inhibited pre-rRNA processing. As a consequence, mutant and depleted cells showed decreased levels of polysomes compared to wild-type cells. Indirect immunofluorescence indicated a predominant localization of Lcp5p in the nucleolus. In addition, antibodies directed against Lcp5p specifically immunoprecipitated the yeast U3 snoRNA snR17, suggesting that the protein is directly involved in pre-rRNA processing.     

Novel interactions of Saccharomyces cerevisiae type 1 protein phosphatase identified by single-step affinity purification and mass spectrometry

The catalytic subunit of Saccharomyces cerevisiae type 1 protein phosphatase (PP1(C)) is encoded by the essential gene GLC7 and is involved in regulating diverse cellular processes. To identify potential regulatory or targeting subunits of yeast PP1(C), we tagged Glc7p at its amino terminus with protein A and affinity-purified Glc7p protein complexes from yeast. The purified proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and identified by peptide mass fingerprint analysis using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. To confirm the accuracy of our identifications, peptides from some of the proteins were also sequenced using high-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry. Only four of the Glc7p-associated proteins that we identified (Mhp1p, Bni4p, Ref2p, and Sds22p) have previously been shown to interact with Glc7p, and multiple components of the CPF (cleavage and polyadenylation factor) complex involved in messenger RNA 3'-end processing were present as major components in the Glc7p-associated protein fraction. To confirm the interaction of Glc7p with this complex, we used the same approach to purify and characterize the components of the yeast CPF complex using protein A-tagged Pta1p. Six known components of the yeast (CPF) complex, together with Glc7p, were identified among the Pta1p-associated polypeptides using peptide mass fingerprint analysis. Thus Glc7p is a novel component of the CPF complex and may therefore be involved regulating mRNA 3'-end processing.     

Yeast mRNA Poly(A) tail length control can be reconstituted in vitro in the absence of Pab1p-dependent Poly(A) nuclease activity

Regulation of poly(A) tail length during mRNA 3'-end formation requires a specific poly(A)-binding protein in addition to the cleavage/polyadenylation machinery. The mechanism that controls polyadenylation in mammals is well understood and involves the nuclear poly(A)-binding protein PABPN1. In contrast, poly(A) tail length regulation is poorly understood in yeast. Previous studies have suggested that the major cytoplasmic poly(A)-binding protein Pab1p acts as a length control factor in conjunction with the Pab1p-dependent poly(A) nuclease PAN, to regulate poly(A) tail length in an mRNA specific manner. In contrast, we recently showed that Nab2p regulates polyadenylation during de novo synthesis, and its nuclear location is more consistent with a role in 3'-end processing than that of cytoplasmic Pab1p. Here, we investigate whether PAN activity is required for de novo poly(A) tail synthesis. Components required for mRNA 3'-end formation were purified from wild-type and pan mutant cells. In both situations, 3'-end formation could be reconstituted whether Nab2p or Pab1p was used as the poly(A) tail length control factor. However, polyadenylation was more efficient and physiologically more relevant in the presence of Nab2p as opposed to Pab1p. Moreover, cell immunofluorescence studies confirmed that PAN subunits are localized in the cytoplasm which suggests that cytoplasmic Pab1p and PAN may act at a later stage in mRNA metabolism. Based on these findings, we propose that Nab2p is necessary and sufficient to regulate poly(A) tail length during de novo synthesis in yeast.     

The WD-repeat protein pfs2p bridges two essential factors within the yeast pre-mRNA 3'-end-processing complex

In the yeast Saccharomyces cerevisiae, pre-mRNA 3'-end processing requires six factors: cleavage factor IA (CF IA), cleavage factor IB (CF IB), cleavage factor II (CF II), polyadenylation factor I (PF I), poly(A) polymerase (Pap1p) and poly(A)-binding protein I (Pab1p). We report the characterization of Pfs2p, a WD-repeat protein previously identified in a multiprotein complex carrying PF I-Pap1p activity. The 3'-end-processing defects of pfs2 mutant strains and the results of immunodepletion and immunoinactivation experiments indicate an essential function for Pfs2p in cleavage and polyadenylation. With a one-step affinity purification method that exploits protein A-tagged Pfs2p, we showed that this protein is part of a CF II-PF I complex. Pull-down experiments with GST fusion proteins revealed direct interactions of Pfs2p with subunits of CF II-PF I and CF IA. These results show that Pfs2p plays an essential role in 3'-end formation by bridging different processing factors and thereby promoting the assembly of the processing complex.     

The role of the Brr5/Ysh1 C-terminal domain and its homolog Syc1 in mRNA 3'-end processing in Saccharomyces cerevisiae

The cleavage/polyadenylation factor (CPF) of Saccharomyces cerevisiae is thought to provide the catalytic activities of the mRNA 3'-end processing machinery, which include endonucleolytic cleavage at the poly(A) site, followed by synthesis of an adenosine polymer onto the new 3'-end by the CPF subunit Pap1. Because of similarity to other nucleases in the metallo-beta-lactamase family, the Brr5/Ysh1 subunit has been proposed to be the endonuclease. The C-terminal domain of Brr5 lies outside of beta-lactamase homology, and its function has not been elucidated. We show here that this region of Brr5 is necessary for cell viability and mRNA 3'-end processing. It is highly homologous to another CPF subunit, Syc1. Syc1 is not essential, but its removal improves the growth of other processing mutants at restrictive temperatures and restores in vitro processing activity to cleavage/ polyadenylation-defective brr5-1 extract. Our findings suggest that Syc1, by mimicking the essential Brr5 C-terminus, serves as a negative regulator of mRNA 3'-end formation.     

Analysis of adenovirus type 2 L1 RNA 3''-end formation in vivo and in vitro.

Downstream sequence requirements for efficient cleavage and polyadenylation at the adenovirus type 2 L1 poly(A) site were determined in vivo in 293 cells and in vitro by using RNA precursors in HeLa cell nuclear extracts. The two cleavage sites used were found to differ in sensitivity to 3'-end deletion in vivo and in vitro.     

SARS coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mRNAs: viral mRNAs are resistant to nsp1-induced RNA cleavage

SARS coronavirus (SCoV) nonstructural protein (nsp) 1, a potent inhibitor of host gene expression, possesses a unique mode of action: it binds to 40S ribosomes to inactivate their translation functions and induces host mRNA degradation. Our previous study demonstrated that nsp1 induces RNA modification near the 5'-end of a reporter mRNA having a short 5' untranslated region and RNA cleavage in the encephalomyocarditis virus internal ribosome entry site (IRES) region of a dicistronic RNA template, but not in those IRES elements from hepatitis C or cricket paralysis viruses. By using primarily cell-free, in vitro translation systems, the present study revealed that the nsp1 induced endonucleolytic RNA cleavage mainly near the 5' untranslated region of capped mRNA templates. Experiments using dicistronic mRNAs carrying different IRESes showed that nsp1 induced endonucleolytic RNA cleavage within the ribosome loading region of type I and type II picornavirus IRES elements, but not that of classical swine fever virus IRES, which is characterized as a hepatitis C virus-like IRES. The nsp1-induced RNA cleavage of template mRNAs exhibited no apparent preference for a specific nucleotide sequence at the RNA cleavage sites. Remarkably, SCoV mRNAs, which have a 5' cap structure and 3' poly A tail like those of typical host mRNAs, were not susceptible to nsp1-mediated RNA cleavage and importantly, the presence of the 5'-end leader sequence protected the SCoV mRNAs from nsp1-induced endonucleolytic RNA cleavage. The escape of viral mRNAs from nsp1-induced RNA cleavage may be an important strategy by which the virus circumvents the action of nsp1 leading to the efficient accumulation of viral mRNAs and viral proteins during infection.     

Characterization of nuclear polyadenylated RNA-binding proteins in Saccharomyces cerevisiae

To study the functions of heterogeneous nuclear ribonucleoproteins (hnRNPs), we have characterized nuclear polyadenylated RNA-binding (Nab) proteins from Saccharomyces cerevisiae. Nab1p, Nab2p, and Nab3p were isolated by a method which uses UV light to cross-link proteins directly bound to poly(A)+ RNA in vivo. We have previously characterized Nab2p, and demonstrated that it is structurally related to human hnRNPs. Here we report that Nab1p is identical to the Np13p/Nop3p protein recently implicated in both nucleocytoplasmic protein shuttling and pre-rRNA processing, and characterize a new nuclear polyadenylated RNA-binding protein, Nab3p. The intranuclear distributions of the Nab proteins were analyzed by three-dimensional immunofluorescence optical microscopy. All three Nab proteins are predominantly localized within the nucleoplasm in a pattern similar to the distribution of hnRNPs in human cells. The NAB3 gene is essential for cell viability and encodes an acidic ribonucleoprotein. Loss of Nab3p by growth of a GAL::nab3 mutant strain in glucose results in a decrease in the amount of mature ACT1, CYH2, and TPI1 mRNAs, a concomitant accumulation of unspliced ACT1 pre-mRNA, and an increase in the ratio of unspliced CYH2 pre-mRNA to mRNA. These results suggest that the Nab proteins may be required for packaging pre-mRNAs into ribonucleoprotein structures amenable to efficient nuclear RNA processing.     

A novel plant in vitro assay system for pre-mRNA cleavage during 3'-end formation

Messenger RNA (mRNA) maturation in eukaryotic cells requires the formation of the 3' end, which includes two tightly coupled steps: the committing cleavage reaction that requires both correct cis-element signals and cleavage complex formation, and the polyadenylation step that adds a polyadenosine [poly(A)] tract to the newly generated 3' end. An in vitro biochemical assay plays a critical role in studying this process. The lack of such an assay system in plants hampered the study of plant mRNA 3'-end formation for the last two decades. To address this, we have now established and characterized a plant in vitro cleavage assay system, in which nuclear protein extracts from Arabidopsis (Arabidopsis thaliana) suspension cell cultures can accurately cleave different pre-mRNAs at expected in vivo authenticated poly(A) sites. The specific activity is dependent on appropriate cis-elements on the substrate RNA. When complemented by yeast (Saccharomyces cerevisiae) poly(A) polymerase, about 150-nucleotide poly(A) tracts were added specifically to the newly cleaved 3' ends in a cooperative manner. The reconstituted polyadenylation reaction is indicative that authentic cleavage products were generated. Our results not only provide a novel plant pre-mRNA cleavage assay system, but also suggest a cross-kingdom functional complementation of yeast poly(A) polymerase in a plant system.