Françoise Wyers

Cryptic Pol II Transcripts Are Degraded by a Nuclear Quality Control Pathway Involving a New Poly(A) Polymerase

Since detection of an RNA molecule is the major criterion to define transcriptional activity, the fraction of the genome that is expressed is generally considered to parallel the complexity of the transcriptome. We show here that several supposedly silent intergenic regions in the genome of S. cerevisiae are actually transcribed by RNA polymerase II, suggesting that the expressed fraction of the genome is higher than anticipated. Surprisingly, however, RNAs originating from these regions are rapidly degraded by the combined action of the exosome and a new poly(A) polymerase activity that is defined by the Trf4 protein and one of two RNA binding proteins, Air1p or Air2p. We show that such a polyadenylation-assisted degradation mechanism is also responsible for the degradation of several Pol I and Pol III transcripts. Our data strongly support the existence of a posttranscriptional quality control mechanism limiting inappropriate expression of genetic information.

Deletion of the PAT1 Gene Affects Translation Initiation and Suppresses a PAB1 Gene Deletion in Yeast

ABSTRACT The yeast poly(A) binding protein Pab1p mediates the interactions between the 5′ cap structure and the 3′ poly(A) tail of mRNA, whose structures synergistically activate translation in vivo and in vitro. We found that deletion of the PAT1 (YCR077c) gene suppresses a PAB1 gene deletion and that Pat1p is required for the normal initiation of translation. A fraction of Pat1p cosediments with free 40S ribosomal subunits on sucrose gradients. ThePAT1 gene is not essential for viability, although disruption of the gene severely impairs translation initiation in vivo, resulting in the accumulation of 80S ribosomes and in a large decrease in the amounts of heavier polysomes. Pat1p contributes to the efficiency of translation in a yeast cell-free system. However, the synergy between the cap structure and the poly(A) tail is maintained in vitro in the absence of Pat1p. Analysis of translation initiation intermediates on gradients indicates that Pat1p acts at a step before or during the recruitment of the 40S ribosomal subunit by the mRNA, a step which may be independent of that involving Pab1p. We conclude that Pat1p is a new factor involved in protein synthesis and that Pat1p might be required for promoting the formation or the stabilization of the preinitiation translation complexes.

Pti1p and Ref2p found in association with the mRNA 3′ end formation complex direct snoRNA maturation

Eukaryotic RNA polymerase II transcribes precursors of mRNAs and of non‐protein‐coding RNAs such as snRNAs and snoRNAs. These RNAs have to be processed at their 3′ ends to be functional. mRNAs are matured by cleavage and polyadenylation that require a well‐characterized protein complex. Small RNAs are also subject to 3′ end cleavage but are not polyadenylated. Here we show that two newly identified proteins, Pti1p and Ref2p, although they were found associated with the pre‐mRNA 3′ end processing complex, are essential for yeast snoRNA 3′ end maturation. We also provide evidence that Pti1p probably acts by uncoupling cleavage and polyadenylation, and functions in coordination with the Nrd1p‐dependent pathway for 3′ end formation of non‐polyadenylated transcripts.

Mpe1, a zinc knuckle protein, is an essential component of yeast cleavage and polyadenylation factor required for the cleavage and polyadenylation of mRNA.

ABSTRACT 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 ofACT1 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.

Association of RNase H activity with yeast RNA polymerase A

EUKARYOTIC RNA polymerases were isolated as large multimeric protein complexes containing two high molecular weight subunits and a collection of smaller polypeptide chains1–3. This structural complexity suggests a multifunctional system, organised around a core enzyme combined with specificity determinants and possibly other proteins involved in regulation of chromatin activity. The purpose of this report is to describe the association of a ribonuclease H activity with yeast RNA polymerase A. This nuclease activity, which specifically degrades RNA–DNA hybrids, is inseparable from the polymerase by various fractionation procedures.

Vesicular Stomatitis Virus Growth in Drosophila melanogaster Cells. II. Modifications of Viral Protein Phosphorylation

The phosphoproteins of vesicular stomatitis virus released from infected Drosophila melanogaster cells were examined. The membrane (M) protein was more phosphorylated than after multiplication in chicken embryo cells, even in Drosophila cell cytoplasm before its association with cellular membranes. Analysis of phosphopeptides generated after partial proteolysis and of phosphoamino acids obtained after complete acid hydrolysis showed that M phosphorylation was quantitatively and qualitatively changed, while NS protein phosphorylation was only slightly modified.

Sigma virus: growth in Drosophila melanogaster cell culture; purification; protein composition and localization

Summary The growth cycle of sigma virus in Drosophila melanogaster cells was studied: optimal virus production was reached about 40 h after infection; virus release declined thereafter and then remained approximately constant (carrier state). In the presence of DEAE-dextran during virus adsorption, more cells became infected and sigma virus production was enhanced. Sigma virus was partially purified by a gentle procedure. Five presumptive virus-specific proteins with molecular weights 210K, 68K, 57K, 44K and 25K were observed. The p68 polypeptide was glycosylated and formed the spikes of the virion particles. The nucleocapsid contained a single major protein, p44, and one or two minor proteins (p210 and probably p57); another protein, p25, was more loosely associated with the nucleocapsid. None of these proteins was found to be phosphorylated.

Yeast RNA Polymerases

INTRODUCTION The presence of multiple forms of RNA polymerases has been recognized in all eukaryotic cells, including plant, insect, fungi and yeast cells (Chambon 1975). A basic, generally accepted assumption is that these related enzymes are specifically synthesizing the various classes of RNA (Zylber and Penman 1971; Reeder and Roeder 1972; Weinmann and Roeder 1974; Chambon 1975). In animal cells, the α -amanitin-sensitive class B (or II) RNA polymerase is believed to synthesize precursor messenger RNA; the moderately sensitive class C (or III) enzyme is responsible for the synthesis of 5S and tRNA; whereas class A (or I) enzyme, which is insensitive to high concentrations of the toxic peptide, probably makes ribosomal RNA. Although it is still premature to extend this conclusion to lower eukaryotic cells, such as yeast cells, the striking similarity between enzymes isolated from various types of cells strongly supports this general hypothesis. One important problem, then, is to understand what determines, on a structural basis, the different specificities of the three classes of RNA polymerase. Additional complexity arises from the presence of subclasses of RNA polymerases. For instance, RNA polymerase B can be resolved into two or three forms of enzyme, B0 (II 0 ), BI (II A ) and BII (II B ), which differ only in the molecular weight of their largest subunit (Chambon 1975; Link and Richter 1975; Schwartz and Roeder 1975). Two forms of RNA polymerase C can also be separated (Schwartz et al. 1974). The in vivo significance of this multiplicity is unclear. Therefore the origin and possible...

Restricted Expression of Viral Glycoprotein in Vesicular Stomatitis Virus-infected Drosophila melanogaster Cells

Vesicular stomatitis virus (VSV) establishes a non-cytopathic persistent infection in Drosophila melanogaster cells. The synthesis of the viral glycoprotein G was specifically inhibited during a post-transcriptional step, whereas the synthesis and turnover of its mRNA were not modified compared with the other viral mRNAs. Another viral glycoprotein, migrating slightly faster than G protein on an SDS-polyacrylamide gel, was detected in infected Drosophila cells. This protein showed most of the characteristics of the intracellular Gs protein found in infected vertebrate cells. The amounts of G protein integrated into mature virions and of soluble Gs protein secreted into the culture medium were reduced greatly during VSV infection in Drosophila cells.