Sequence requirements of the bidirectional yeast TRP4 mRNA 3'-end formation signal.

The yeast TRP4 3'-end formation signal functions in both orientations in an in vivo test system. We show here that the TRP4 3'-end formation element consists of two functionally different sequence regions. One region of approximately 70 nucleotides is located in the untranslated region between the translational stop codon and the major poly(A) site. The major poly(A) site is not part of this region and can be deleted without a decrease in TRP4 3'-end formation. 5'and 3'deletions and point mutations within this region affected 3'-end formation similarly in both orientations. In the center of this region the motif TAGT is located on the antisense strand. Point mutations within this motif resulted in a drastic reduce of 3'-end formation activity in both orientations. A second region consists of the 3'-end of the TRP4 open reading frame and is required for 3'-end formation in forward orientation. A single point mutation in a TAGT motif of the TRP4 open reading frame abolished TRP4 mRNA 3'-end formation in forward orientation and had no effect on the reverse orientation.     

REF2 encodes an RNA-binding protein directly involved in yeast mRNA 3'-end formation.

The Saccharomyces cerevisiae mutant ref2-1 (REF = RNA end formation) was originally identified by a genetic strategy predicted to detect decreases in the use of a CYC1 poly(A) site interposed within the intron of an ACT1-HIS4 fusion reporter gene. Direct RNA analysis now proves this effect and also demonstrates the trans action of the REF2 gene product on cryptic poly(A) sites located within the coding region of a plasmid-borne ACT1-lacZ gene. Despite impaired growth of ref2 strains, possibly because of a general defect in the efficiency of mRNA 3'-end processing, the steady-state characteristics of a variety of normal cellular mRNAs remain unaffected. Sequencing of the complementing gene predicts the Ref2p product to be a novel, basic protein of 429 amino acids (M(r), 48,000) with a high-level lysine/serine content and some unusual features. Analysis in vitro, with a number of defined RNA substrates, confirms that efficient use of weak poly(A) sites requires Ref2p: endonucleolytic cleavage is carried out accurately but at significantly lower rates in extracts prepared from delta ref2 cells. The addition of purified, epitope-tagged Ref2p (Ref2pF) reestablishes wild-type levels of activity in these extracts, demonstrating direct involvement of this protein in the cleavage step of 3' mRNA processing. Together with the RNA-binding characteristics of Ref2pF in vitro, our results support an important contributing role for the REF2 locus in 3'-end processing. As the first gene genetically identified to participate in mRNA 3'-end maturation prior to the final polyadenylation step, REF2 provides an ideal starting point for identifying related genes in this event.     

Arginine methylation and binding of Hrp1p to the efficiency element for mRNA 3'-end formation

Hrp1p is a heterogeneous ribonucleoprotein (hnRNP) from the yeast Saccharomyces cerevisiae that is involved in the cleavage and polyadenylation of the 3'-end of mRNAs and mRNA export. In addition, Hrplp is one of several RNA-binding proteins that are posttranslationally modified by methylation at arginine residues. By using functional recombinant Hrp1p, we have identified RNA sequences with specific high affinity binding sites. These sites correspond to the efficiency element for mRNA 3'-end formation, UAUAUA. To examine the effect of methylation on specific RNA binding, purified recombinant arginine methyltransferase (Hmt1p) was used to methylate Hrp1p. Methylated Hrp1p binds with the same affinity to UAUAUA-containing RNAs as unmethylated Hrpl p indicating that methylation does not affect specific RNA binding. However, RNA itself inhibits the methylation of Hrp1p and this inhibition is enhanced by RNAs that specifically bind Hrpl p. Taken together, these data support a model in which protein methylation occurs prior to protein-RNA binding in the nucleus.     

Homologous mRNA 3'' end formation in fission and budding yeast.

Sequences resembling polyadenylation signals of higher eukaryotes are present downstream of the Schizosaccharomyces pombe ura4+ and cdc10+ coding regions and function in HeLa cells. However, these and other mammalian polyadenylation signals are inactive in S. pombe. Instead, we find that polyadenylation signals of the CYC1 gene of budding yeast Saccharomyces cerevisiae function accurately and efficiently in fission yeast. Furthermore, a 38 bp deletion which renders this RNA processing signal non-functional in S. cerevisiae has the equivalent effect in S. pombe. We demonstrate that synthetic pre-mRNAs encoding polyadenylation sites of S. pombe genes are accurately cleaved and polyadenylated in whole cell extracts of S. cerevisiae. Finally, as is the case in S. cerevisiae, DNA sequences encoding regions proximal to the S. pombe mRNA 3' ends are found to be extremely AT rich; however, no general sequence motif can be found. We conclude that although fission yeast has many genetic features in common with higher eukaryotes, mRNA 3' end formation is significantly different and appears to be formed by an RNA processing mechanism homologous to that of budding yeast. Since fission and budding yeast are evolutionarily divergent, this lower eukaryotic mechanism of mRNA 3' end formation may be generally conserved.     

5'-end formation of yeast 5.8SL rRNA is an endonucleolytic event

Like most eukaryotes, Saccharomyces cerevisiae cells contain a minor 5.8SL rRNA that, relative to the major 5.8SS species, carries several extra nucleotides at the 5'-end. The two species are produced by alternative pathways that differ in the events removing the 3'-terminal region of Internal Transcribed Spacer 1 from the 27SA2 pre-rRNA. Whereas the pathway leading to 5.8SS rRNA is well established, that producing the 5'-end of 5.8SL (called B1L) is poorly understood. Northern analysis of two different mutants of S. cerevisiae that overproduce 5.8SL rRNA revealed the presence of a fragment corresponding to the 3'-terminal region of Internal Transcribed Spacer 1 (ITS1) directly upstream from site B1L. Immunoprecipitation experiments showed this fragment to be associated with the trans-acting factor Rrp5p required for processing at the early sites A0-A3. Together these data clearly support that the 5'-end of 5.8SL rRNA is an endonucleolytic event. In vivo mutational analysis demonstrated the lack of any cis-acting sequence elements directing this cleavage within ITS1.     

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.     

3'-end processing of the maize 27 kDa zein mRNA

Cis -regulatory elements involved in the mRNA 3'-end processing of the 27 kDa zein gene have been investigated by deletion and site-directed mutagenesis analyses. In the 3' flanking region of the 27 kDa zein gene, several AATAAA-like sequences and a sequence resembling the mammalian GT-rich sequence are present around the polyadenylation sites. Among the multiple AATAAA-like sequences, the duplicated AATGAA motifs, located 30–40 bp upstream from the polyadenylation sites, have been shown to play roles as polyadenylation signals. Although either of the two AATGAA motifs can function as a polyadenylation signal in chimeric gene constructs, the one proximal to the polyadenylation sites is likely to be the functional polyadenylation signal in the 27 kDa zein gene. Deletion of the downstream GT-rich sequence as well as alteration of the sequence surrounding the poly-adenylation sites has little effect on the mRNA 3'-end processing. However, the sequence elements located upstream from the polyadenylation signals are essential for the mRNA 3'-end processing. Mutations in the AATGAA motifs or the upstream sequences reduced the level of a reporter gene expression. A model depicting the mechanism involved in the 3'-end processing of the 27 kDa zein mRNA is presented.     

3'-end labeling of RNA with recombinant yeast poly(A) polymerase.

Two commonly used methods to end-label RNA-molecules are 5'-end labeling by polynucleotide kinase and 3'-end labeling with pCp and T4 RNA ligase. We show here that RNA 3'-ends can also be labeled with the chain-terminating analogue cordycepin 5'-triphosphate (3'-deoxy-ATP) which is added by poly(A) polymerase. For a synthetic RNA it is shown that 40% of cordycepin becomes incorporated when the nucleotide is used at limiting concentrations and that with an excess of cordycepin 5'-triphosphate essentially all the RNA becomes modified at its 3'-end. The reaction is complete within minutes and the RNA product is uniform and suitable for sequence analysis. The efficiency of labeling varies with different RNA-molecules and is different from RNA ligase. Poly(A) polymerase preferentially labels longer RNA-molecules whereas short RNA-molecules are labeled more efficiently by T4 RNA ligase.     

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.