Pbp1p, a factor interacting with Saccharomyces cerevisiae poly(A)-binding protein, regulates polyadenylation

The poly(A) tail of an mRNA is believed to influence the initiation of translation, and the rate at which the poly(A) tail is removed is thought to determine how fast an mRNA is degraded. One key factor associated with this 3'-end structure is the poly(A)-binding protein (Pab1p) encoded by the PAB1 gene in Saccharomyces cerevisiae. In an effort to learn more about the functional role of this protein, we used a two-hybrid screen to determine the factor(s) with which it interacts. We identified five genes encoding factors that specifically interact with the carboxy terminus of Pab1p. Of a total of 44 specific clones identified, PBP1 (for Pab1p-binding protein) was isolated 38 times. Of the putative interacting genes examined, PBP1 promoted the highest level of resistance to 3-aminotriazole (>100 mM) in constructs in which HIS3 was used as a reporter. We determined that a fraction of Pbp1p cosediments with polysomes in sucrose gradients and that its distribution is very similar to that of Pab1p. Disruption of PBP1 showed that it is not essential for viability but can suppress the lethality associated with a PAB1 deletion. The suppression of pab1Delta by pbp1Delta appears to be different from that mediated by other pab1 suppressors, since disruption of PBP1 does not alter translation rates, affect accumulation of ribosomal subunits, change mRNA poly(A) tail lengths, or result in a defect in mRNA decay. Rather, Pbp1p appears to function in the nucleus to promote proper polyadenylation. In the absence of Pbp1p, 3' termini of pre-mRNAs are properly cleaved but lack full-length poly(A) tails. These effects suggest that Pbp1p may act to repress the ability of Pab1p to negatively regulate polyadenylation.     

Control of cleavage site selection during mRNA 3' end formation by a yeast hnRNP

Endonucleolytic cleavage of pre-mRNAs is the first step during eukaryotic mRNA 3' end formation. It has been proposed that cleavage factors CF IA, CF IB and CF II are required for pre-mRNA 3' end cleavage in yeast. CF IB is composed of a single polypeptide, Nab4p/Hrp1p, which is related to the A/B group of metazoan heterogeneous nuclear ribonucleoproteins (hnRNPs) that function as antagonistic regulators of 5' splice site selection. Here, we provide evidence that Nab4p/Hrp1p is not required for pre-mRNA 3' end endonucleolytic cleavage. We show that CF IA and CF II devoid of Nab4p/Hrp1p are sufficient to cleave a variety of RNA substrates but that cleavage occurs at multiple sites. Addition of Nab4p/Hrp1p prevents these alternative cleavages in a concentration-dependent manner, suggesting an essential and conserved role for some hnRNPs in pre-mRNA cleavage site selection.     

Regulation of tRNA suppressor activity by an intron-encoded polyadenylation signal

A 26-nt sequence from the 3' UTR of the yeast GAL7 mRNA directs accurate and efficient cleavage and polyadenylation to form the 3' end of the GAL7 mRNA in vivo and in vitro. Here we asked whether this polyadenylation signal can function within the context of a tRNA. Insertion of the GAL7 signal into the intron of the dominant SUP4 nonsense suppressor allowed us to judge the effect of the insert on SUP4 function by observation of nonsense suppression efficiency in vivo. The GAL7 signal impairs the function of SUP4 in an orientation-dependent manner in vivo, consistent with its ability to specify cleavage and polyadenylation in this context in vitro. Mutation of a UA repeat within the GAL7 signal restores SUP4 function partially, consistent with the role of this repeat as an efficiency element in polyadenylation. Mutations that impair the mRNA 3' end-processing factors Rna14p and Rna15p restore suppressor function partially. Northern blot analysis, PCR amplification, and DNA sequence analysis show that the GAL7 signal directs polyadenylation within the body of pre-SUP4 and within the terminator, suggesting that polyadenylation inhibits 5' and 3' end processing, as well as removal of the pre-tRNA intron. These findings indicate that the GAL7 polyadenylation signal is capable of targeting a pre-tRNA to the mRNA processing pathway.     

Poly(A)-tail-promoted translation in yeast: implications for translational control

The cap structure and the poly(A) tail synergistically activate mRNA translation in vivo. Recent work using Saccharomyces cerevisiae spheroplasts and a yeast cell-free translation system revealed that the poly(A) tail can function as an independent promotor for ribosome recruitment, to internal initiation sites within an mRNA. This raises the question of how regulatory upstream open reading frames and translational repressor proteins binding to the 5'UTR can function, as well as how regulated polyadenylation can support faithful activation of protein synthesis. We investigated the function of the regulatory upstream open reading frame 4 from the yeast GCN 4 gene and the effect of IRP-1 binding to an iron-responsive element introduced into the 5' UTR of reporter mRNAs. Both manipulations effectively block cap-dependent translation, whereas ribosome recruitment promoted by the poly(A) tail under non-competitive conditions can efficiently bypass both blocks. We show that the synergistic use of both, the cap structure and the poly-A tail enforced by mRNA competition reinstates the full extent of translational control by both types of 5' UTR regulatory elements. With a view towards regulated polyadenylation, we studied the function of poly(A) tails of defined length on the translation of capped mRNAs. We find that poly(A) tail elongation increases translational efficiency, particularly under competitive conditions. Our results integrate recent findings on the function of the poly(A) tail into an understanding of translational control.     

A dependent pathway of cytoplasmic polyadenylation reactions linked to cell cycle control by c-mos and CDK1 activation

During oocyte maturation and early development, mRNAs receive poly(A) in the cytoplasm at distinct times relative to one another and to the cell cycle. These cytoplasmic polyadenylation reactions do not occur during oogenesis, but begin during oocyte maturation and continue throughout early development. In this report, we focus on the link between cytoplasmic polyadenylation and control of the cell cycle during meiotic maturation. Activation of maturation promoting factor, a complex of CDK1 and cyclin, is required for maturation and dependent on c-mos protein kinase. We demonstrate here that two classes of polyadenylation exist during oocyte maturation, defined by their dependence of c-mos and CDK1 protein kinases. Polyadenylation of the first class of mRNAs (class I) is independent of c-mos and CDK1 kinase activities, whereas polyadenylation of the second class (class II) requires both of these activities. Class I polyadenylation, through its effects on c-mos mRNA, is required for class II polyadenylation. cis-acting elements responsible for this distinction reside in the 3'-untranslated region, upstream of the polyadenylation signal AAUAAA. Cytoplasmic polyadenylation elements (CPEs) are sufficient to specify class I polyadenylation, and subtle changes in the CPE can substantially, though not entirely, shift an RNA from class I to class II. Activation of class I polyadenylation events is independent of hyperphosphorylation of CPE-binding protein or poly(A) polymerase, and requires cellular protein synthesis. The two classes of polyadenylation and of mRNA define a dependent pathway, in which polyadenylation of certain mRNAs requires the prior polyadenylation of another. We propose that this provides one method of regulating the temporal order of polyadenylation events, and links polyadenylation to the control of the meiotic cell cycle.     

Sequence similarity between the 73-kilodalton protein of mammalian CPSF and a subunit of yeast polyadenylation factor I

The 3' ends of most eukaryotic messenger RNAs are generated by endonucleolytic cleavage and polyadenylation. In mammals, the cleavage and polyadenylation specificity factor (CPSF) plays a central role in both steps of the processing reaction. Here, the cloning of the 73-kilodalton subunit of CPSF is reported. Sequence analyses revealed that a yeast protein (Ysh1) was highly similar to the 73-kD polypeptide. Ysh1 constitutes a new subunit of polyadenylation factor I (PFI), which has a role in yeast pre-mRNA 3'-end formation. This finding was unexpected because in contrast to CPSF, PFI is only required for the polyadenylation reaction. These results contribute to the understanding of how 3'-end processing factors may have evolved.     

Creatine phosphate, not ATP, is required for 3' end cleavage of mammalian pre-mRNA in vitro

The poly(A) tail of a mammalian mRNA is generated by endonucleolytic cleavage and poly(A) addition. Previous studies conducted with nuclear extracts suggested an ATP requirement for the cleavage step. We have reexamined the cofactor requirement, initially with the SV40 late pre-mRNA, which requires for cleavage four protein factors, cleavage and polyadenylation specificity factor, cleavage stimulation factor, cleavage factor I, and cleavage factor II. Using highly purified preparations of these factors, which lacked detectable creatine phosphokinase and ATPase activities, creatine phosphate (CP) was, surprisingly, found to be sufficient to promote efficient cleavage. Although other phosphate compounds substituted poorly or not at all for CP, another phosphoguanidine, arginine phosphate, was fully functional. Notably, ATP was neither necessary nor sufficient, and could in fact inhibit the reaction. Treatment of the purified factors with hexokinase plus glucose (to deplete any contaminating ATP) was without effect, as was addition of EDTA. Using 32P-labeled CP, we found that neither hydrolysis of CP nor phosphate transfer from CP occurred during the cleavage reaction. CP also allowed cleavage of the adenovirus 2 L3 pre-mRNA. However, in this case, ATP both enhanced the reaction and influenced the precise site of cleavage, perhaps reflecting the requirement of poly(A) polymerase for cleavage of this RNA. These results indicate that ATP is not essential for 3' pre-mRNA cleavage and that CP or a related compound can function as a necessary cofactor.     

Polyadenylation of stable RNA precursors in vivo

Polyadenylation at the 3' terminus has long been considered a specific feature of mRNA and a few other unstable RNA species. Here we show that stable RNAs in Escherichia coli can be polyadenylated as well. RNA molecules with poly(A) tails are the major products that accumulate for essentially all stable RNA precursors when RNA maturation is slowed because of the absence of processing exoribonucleases; poly(A) tails vary from one to seven residues in length. The polyadenylation process depends on the presence of poly(A) polymerase I. A stochastic competition between the exoribonucleases and poly(A) polymerase is proposed to explain the accumulation of polyadenylated RNAs. These data indicate that polyadenylation is not unique to mRNA, and its widespread occurrence suggests that it serves a more general function in RNA metabolism.     

Modifications of the 5' cap of mRNAs during Xenopus oocyte maturation: independence from changes in poly(A) length and impact on translation

The translation of specific maternal mRNAs is regulated during early development. For some mRNAs, an increase in translational activity is correlated with cytoplasmic extension of their poly(A) tails; for others, translational inactivation is correlated with removal of their poly(A) tails. Recent results in several systems suggest that events at the 3' end of the mRNA can affect the state of the 5' cap structure, m7G(5')ppp(5')G. We focus here on the potential role of cap modifications on translation during early development and on the question of whether any such modifications are dependent on cytoplasmic poly(A) addition or removal. To do so, we injected synthetic RNAs into Xenopus oocytes and examined their cap structures and translational activities during meiotic maturation. We draw four main conclusions. First, the activity of a cytoplasmic guanine-7-methyltransferase increases during oocyte maturation and stimulates translation of an injected mRNA bearing a nonmethylated GpppG cap. The importance of the cap for translation in oocytes is corroborated by the sensitivity of protein synthesis to cap analogs and by the inefficient translation of mRNAs bearing nonphysiologically capped 5' termini. Second, deadenylation during oocyte maturation does not cause decapping, in contrast to deadenylation-triggered decapping in Saccharomyces cerevisiae. Third, the poly(A) tail and the N-7 methyl group of the cap stimulate translation synergistically during oocyte maturation. Fourth, cap ribose methylation of certain mRNAs is very inefficient and is not required for their translational recruitment by poly(A). These results demonstrate that polyadenylation can cause translational recruitment independent of ribose methylation. We propose that polyadenylation enhances translation through at least two mechanisms that are distinguished by their dependence on ribose modification.     

DSEF-1 is a member of the hnRNP H family of RNA-binding proteins and stimulates pre-mRNA cleavage and polyadenylation in vitro

DSEF-1 protein selectively binds to a G-rich auxiliary sequence element which influences the efficiency of processing of the SV40 late polyadenylation signal. We have obtained cDNA clones of DSEF-1 using sequence information from tryptic peptides isolated from DSEF-1 protein purified from HeLa cells. DSEF-1 protein contains three RNA-binding motifs and is a member of the hnRNP H family of RNA-binding proteins. Recombinant DSEF-1 protein stimulated the efficiency of cleavage and polyadenylation in an AAUAAA-dependent manner in in vitro reconstitution assays. DSEF-1 protein was shown to be able to interact with several poly(A) signals that lacked a G-rich binding site using a less stringent, low ionic strength gel band shift assay. Recombinant DSEF-1 protein specifically stimulated the processing of all of the poly(A) signals tested that contained a high affinity G-rich or low affinity binding site. DSEF-1 specifically increased the level of cross-linking of the 64 kDa protein of CstF to polyadenylation substrate RNAs. These observations suggest that DSEF-1 is an auxiliary factor that assists in the assembly of the general 3'-end processing factors onto the core elements of the polyadenylation signal.     

Yeast heat shock mRNAs are exported through a distinct pathway defined by Rip1p

We reported previously that heat or ethanol shock in Saccharomyces cerevisiae leads to nuclear retention of most poly(A)+ RNA but heat shock mRNAs (encoding Hsp70 proteins Ssa1p and Ssa4p) are efficiently exported in a process that is independent of the small GTPase Ran/Gsp1p, which is essential for most nucleocytoplasmic transport. To gain further insights into proteins essential or nonessential for export of heat shock mRNAs, in situ hybridization analyses to detect mRNA and pulse-labeling of proteins were used to examine several yeast mutant strains for their ability to export heat shock mRNAs following stress. Rip1p is a 42-kD protein associated with nuclear pore complexes and contains nucleoporin-like repeat sequences. It is dispensable for growth of yeast cells under normal conditions, but we report that it is essential for the export of heat shock mRNAs following stress. When SSA4 mRNA was induced from a GAL promoter in the absence of stress, it was efficiently exported in a strain lacking RIP1, indicating that Rip1p is required for export of heat shock mRNAs only following stress. Npl3p, a key mediator of export of poly(A)+ RNA, was not required for heat shock mRNA export, whereas Rss1p/Gle1p, a NES-containing factor essential for poly(A)+ RNA export, was also required for export of heat shock mRNAs after stress. High-level expression of the HIV-1 Rev protein, but not of Rev mutants, led to a partial block in export of heat shock mRNAs following stress. The data suggest a model wherein the requirement for Npl3p defines the mRNA export pathway, the requirement for Rip1p defines a pathway used for export of heat shock mRNAs after stress, and additional factors, including Rss1p/Gle1p and several nucleoporins (Rat7p/Nup159p, Rat2p/Nup120p, and Nup145p/Rat10p), are required in both pathways.     

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.     

RNA structure is a critical determinant of poly(A) site recognition by cleavage and polyadenylation specificity factor

Sequence conservation among mammalian poly(A) sites is limited to the sequence AAUAAA, coupled with an amorphous downstream U- or GU-rich region. Since these sequences may also occur within the coding region of mRNAs, additional information must be required to define authentic poly(A) sites. Several poly(A) sites have been shown to contain sequences outside the core elements that enhance the efficiency of 3' processing in vivo and in vitro. The human immunodeficiency virus type 1, equine infectious anemia virus, and adenovirus L1 3' processing enhancers have been shown to promote the binding of cleavage and polyadenylation specificity factor (CPSF), the factor responsible for recognition of AAUAAA, to the pre-mRNA, thereby facilitating the assembly of a stable 3' processing complex. We have used in vitro selection to examine the mechanism by which the human immunodeficiency virus type 1 3' processing enhancer promotes the interaction of CPSF with the AAUAAA hexamer. Surprisingly, RNAs selected for efficient polyadenylation were related by structure rather than sequence. Therefore, in the absence of extensive sequence conservation, our results strongly suggest that RNA structure is a critical determinant of poly(A) site recognition by CPSF and may play a key role in poly(A) site definition.     

Nucleus-associated pools of Rna1p, the Saccharomyces cerevisiae Ran/TC4 GTPse activating protein involved in nucleus/cytosol transit

Rna1p is the GTPase activating enzyme for Ran/TC4, a Ras-like GTPase necessary for nuclear/cytosolic exchange. Although most wild-type Rna1p is located in the cytosol, we found that the vast majority of the mutant Rna1-1p and, under appropriate physiological conditions, a small portion of the wild-type Rna1p cofractionate with yeast nuclei. Subnuclear fractionation studies show that most of the Rna1p is tightly associated with nuclear components, and that a portion of the active protein can be solubilized by treatments that fail to solubilize inactive Rna1-1p. To learn the precise nuclear locations of the Rna1 proteins, we studied their subcellular distributions in HeLa cells. By indirect immuno-fluorescence we show that wild-type Rna1p has three subcellular locations. The majority of the protein is distributed throughout the cytosol, but a portion of the protein is nucleus-associated, located at both the cytosolic surface and within the nucleoplasm. Mutant Rna1-1p is found at the outer nuclear surface and in the cytosol. We propose that a small pool of the wild-type Rna1p is located in the nuclear interior, supporting the model that the same components of the Ran/TC4 GTPase cycle exist on both sides of the nuclear membrane.     

Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits 3'end formation of cellular pre-mRNAs

Inhibition of the nuclear export of poly(A)-containing mRNAs caused by the influenza A virus NS1 protein requires its effector domain. Here, we demonstrate that the NS1 effector domain functionally interacts with the cellular 30 kDa subunit of CPSF, an essential component of the 3' end processing machinery of cellular pre-mRNAs. In influenza virus-infected cells, the NS1 protein is physically associated with CPSF 30 kDa. Binding of the NS1 protein to the 30 kDa protein in vitro prevents CPSF binding to the RNA substrate and inhibits 3' end cleavage and polyadenylation of host pre-mRNAs. The NS1 protein also inhibits 3' end processing in vivo, and the uncleaved pre-mRNA remains in the nucleus. Via this novel regulation of pre-mRNA 3' end processing, the NS1 protein selectively inhibits the nuclear export of cellular, and not viral, mRNAs.     

Human RanGTPase-activating protein RanGAP1 is a homologue of yeast Rna1p involved in mRNA processing and transport

RanGAP1 is the GTPase activator for the nuclear Ras-related regulatory protein Ran, converting it to the putatively inactive GDP-bound state. Here, we report the amino acid sequence of RanGAP1, derived from cDNA and peptide sequences. We found it to be homologous to murine Fug1, implicated in early embryonic development, and to Rna1p from Saccharomyces cerevisiae and Schizosaccharomyces pombe. Mutations of budding yeast RNA1 are known to result in defects in RNA processing and nucleocytoplasmic mRNA transport. Concurrently, we have isolated Rna1p as the major RanGAP activity from Sc. pombe. Both this protein and recombinant Rna1p were found to stimulate RanGTPase activity to an extent almost identical to that of human RanGAP1, indicating the functional significance of the sequence homology. The Ran-specific guanine nucleotide exchange factor RCC1 and its yeast homologues are restricted to the nucleus, while Rna1p is reported to be localized to the cytoplasm. We suggest a model in which both activities, nuclear GDP-to-GTP exchange on Ran and cytoplasmic hydrolysis of Ran-bound GTP, are essential for shuttling of Ran between the two cellular compartments. Thus, a defect in either of the two antagonistic regulators of Ran would result in a shutdown of Ran-dependent transport processes, in agreement with the almost identical phenotypes described for such defects in budding yeast.     

Participation of the nuclear cap binding complex in pre-mRNA 3' processing

Communication between the 5' and 3' ends is a common feature of several aspects of eukaryotic mRNA metabolism. In the nucleus, the pre-mRNA 5' end is bound by the nuclear cap binding complex (CBC). This RNA-protein complex plays an active role in both splicing and RNA export. We provide evidence for participation of CBC in the processing of the 3' end of the message. Depletion of CBC from HeLa cell nuclear extract strongly reduced the endonucleolytic cleavage step of the cleavage and polyadenylation process. Cleavage was restored by addition of recombinant CBC. CBC depletion was found to reduce the stability of poly(A) site cleavage complexes formed in nuclear extract. We also provide evidence that the communication between the 5' and 3' ends of the pre-mRNA during processing is mediated by the physical association of the CBC/cap complex with 3' processing factors bound at the poly(A) site. These observations, along with previous data on the function of CBC in splicing, illustrate the key role played by CBC in pre-mRNA recognition and processing. The data provides further support for the hypothesis that pre-mRNAs and mRNAs may exist and be functional in the form of "closed-loops," due to interactions between factors bound at their 5' and 3' ends.     

3'-end-forming signals of yeast mRNA

It was previously shown that three distinct but interdependent elements are required for 3' end formation of mRNA in the yeast Saccharomyces cerevisiae: (i) the efficiency element TATATA and related sequences, which function by enhancing the efficiency of positioning elements; (ii) positioning elements, such as TTAAGAAC and AAGAA, which position the poly(A) site; and (iii) the actual site of polyadenylation. In this study, we have shown that several A-rich sequences, including the vertebrate poly(A) signal AATAAA, are also positioning elements. Saturated mutagenesis revealed that optimum sequences of the positioning element were AATAAA and AAAAAA and that this element can tolerate various extents of replacements. However, the GATAAA sequence was completely ineffective. The major cleavage sites determined in vitro corresponded to the major poly(A) sites observed in vivo. Our findings support the assumption that some components of the basic polyadenylation machinery could have been conserved among yeasts, plants, and mammals, although 3' end formation in yeasts is clearly distinct from that of higher eukaryotes.