Distinct cis-acting signals enhance 3'' endpoint formation of CYC1 mRNA in the yeast Saccharomyces cerevisiae.

The cyc1-512 mutant of the yeast Saccharomyces cerevisiae contains a 38 bp deletion in the 3' untranslated region of the CYC1 gene, resulting in CYC1 mRNAs that are elongated, presumably labile, and reduced to 10% of the normal level. Analysis with S1 nuclease and a novel PCR procedure revealed that the low amount of cyc1-512 mRNA contained many discrete 3' termini at certain sites, ranging from the wild-type position to over 2000 nucleotides (nt) downstream. The cyc1-512 mRNA deficiency was completely or almost completely restored in eight intragenic revertants that contained six different single and multiple base-pair changes within a 300 bp region downstream from the translation terminator codon. Two of the six different reversions formed the sequence TAG...TATGTA, whereas the other four reversions created the sequences TATATA or TACATA. The positions of these revertant sequences varied, even though they caused an increased use of specific major downstream mRNA 3' endpoints, apparently identical to those seen in the cyc1-512 mRNA. However, several revertants contained minor end points not corresponding to any of the cyc1-512 mRNAs. The capacity of these three signals to form 3' ends was confirmed with sequences constructed by site-directed mutagenesis. We therefore suggest that the production of 3' termini of yeast mRNA may involve at least two functionally distinct elements working in concert. One type of element determines the sites of preferred 3' mRNA termini, as represented by the cyc1-512 termini. The second type of element, which includes TAG...TATGTA and TATATA motifs, operates at a distance to enhance the use of the downstream 3' preferred sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Different sequence elements are required for function of the cauliflower mosaic virus polyadenylation site in Saccharomyces cerevisiae compared with in plants.

We show that the polyadenylation site derived from the plant cauliflower mosaic virus (CaMV) is specifically functional in the yeast Saccharomyces cerevisiae. The mRNA 3' endpoints were mapped at the same position in yeast cells as in plants, and the CaMV polyadenylation site was recognized in an orientation-dependent manner. Mutational analysis of the CaMV 3'-end-formation signal revealed that multiple elements are essential for proper activity in yeast cells, including two upstream elements that are situated more than 100 and 43 to 51 nucleotides upstream of the poly(A) addition site and the sequences at or near the poly(A) addition site. A comparison of the sequence elements that are essential for proper function of the CaMV signal in yeast cells and plants showed that both organisms require a distal and a proximal upstream element but that these sequence elements are not identical in yeast cells and plants. The key element for functioning of the CaMV signal in yeast cells is the sequence TAGTATGTA, which is similar to a sequence previously proposed to act in yeast cells as a bipartite signal, namely, TAG ... TATGTA. Deletion of this sequence in the CaMV polyadenylation signal abolished 3'-end formation in yeast cells, and a single point mutation in this motif reduced the activity of the CaMV signal to below 15%. These results indicate that the bipartite sequence element acts as a signal for 3'-end formation in yeast cells but only together with other cis-acting elements.     

Recognition of polyadenylation sites in yeast pre-mRNAs by cleavage and polyadenylation factor.

Recognition of poly(A) sites in yeast pre-mRNAs is poorly understood. Employing an in vitro cleavage system with cleavage and polyadenylation factor (CPF) and cleavage factor IA we show that the efficiency and positioning elements are dispensable for poly(A)-site recognition within a short CYC1 substrate in vitro. Instead, U-rich elements immediately upstream and downstream of the poly(A) site mediate cleavage-site recognition within CYC1 and ADH1 pre-mRNAs. These elements act in concert with the poly(A) site to produce multiple recognition sites for the processing machinery, since combinations of mutations within these elements were most effective in cleavage inhibition. Intriguingly, introduction of a U-rich element downstream of the GAL7 poly(A) site strongly enhanced cleavage, underscoring the importance of downstream sequences in general. RNA- binding analyses demonstrate that cleavage depends on the recognition of the poly(A)-site region by CPF. Consistent with in vitro results, mutation of sequences upstream and downstream of the poly(A) site affected 3'-end formation in vivo. A model for yeast pre-mRNA cleavage-site recognition outlines an unanticipated high conservation of yeast and mammalian 3'-end processing mechanisms.     

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.     

An Alu-mediated deletion at Xp11.23 leading to Wiskott-Aldrich syndrome

Wiskott-Aldrich syndrome (WAS) is an X-linked disease characterized by thrombocytopenia, eczema and immunodeficiency of varying severity. The WASP gene, mutations of which are responsible for the phenotype, maps to Xp11.23. We describe here a patient with a large deletion in the Xp11.23 region. The deletion, which totals 15.8 kb, begins downstream of DXS1696 and encompasses 13 kb upstream of WASP and includes the distal and proximal promoters and exons 1-6. Analysis of the 5'-boundary region identified sequences missing in the Human Genome database and, as a result, the normal DNA sequence was revised to include 743 bp of novel sequence (AF466616). The patient's upstream breakpoint was localized to an AluSg element within a highly repetitive DNA region containing other Alu elements. A 26-bp recombinogenic element is located downstream of the 5' breakpoint. A 16-bp sequence just upstream of the 5' breakpoint shares close homology with the sequence that spans the 3' breakpoint in intron 6. A heptanucleotide of unknown origin, CAGGGGG, links the 5' and 3' breakpoints. To our knowledge this is the largest deletion in a WAS patient.     

Analysis of human papillomavirus type 16 late mRNA 3' processing signals in vitro and in vivo.

In the human papillomavirus type 16 genome, three late mRNA putative 3' processing signals, designated LP1, LP2, and LP3, are located downstream of the late coding region. Our results show, both in vitro and in vivo, that in HeLa cells, the LP2 signal functions. Thus, the restriction in human papillomavirus type 16 late-gene expression observed in HeLa cells and other nondifferentiated epithelial cells is not achieved by regulation of late mRNA poly(A) site usage. Interestingly, alteration of three nucleotides in the GU-rich downstream sequence element converts the nonfunctional LP1 to an efficient 3' processing site, suggesting that LP1 may function in cell types other than HeLa, such as differentiated keratinocytes. Our transfection studies have identified a negative regulatory element located immediately upstream of the late mRNA 3' processing signals; this element was not associated with any alteration in 3' processing and may act as an mRNA instability element.     

The DAL7 promoter consists of multiple elements that cooperatively mediate regulation of the gene''s expression.

Expression of the allantoin system genes in Saccharomyces cerevisiae is induced by allophanate or its analog, oxalurate. This work provides evidence for the involvement of distinct types of cis-acting elements in the induction process. The first element was found to have the properties of an upstream activation sequence (UAS). This element was localized to a 16-base-pair (bp) DNA fragment containing a short 5-bp sequence that occurred repeatedly in the upstream region of DAL7. When present in two or more copies, the 16-bp fragment supported high-level beta-galactosidase production in a CYC1-lacZ expression vector; there was, however, no response to the allantoin pathway inducer. The second element had the properties of a negatively acting element or upstream repression sequence (URS). This element was localized to a 16-bp DNA fragment containing an 8-bp sequence that was repeated four times in the upstream region of DAL7. A fragment containing the 8-bp repeated sequence placed adjacent to the UAS-containing fragment mediated inhibition of the ability of the UAS to support lacZ expression regardless of whether inducer was present. A third element, designated an upstream induction sequence (UIS), was required for response to inducer. The UIS was localized to a small DNA fragment containing an approximately 10-bp sequence that was repeated twice in the upstream region of DAL7. When a fragment containing the 10-bp repeated sequence was placed adjacent to these UAS and URS elements, the construction (UIS-UAS-URS) supported normal oxalurate-mediated induction of beta-galactosidase synthesis. These data are consistent with the suggestion that multiple, cis-acting elements participate in the induction process.     

Intercistronic region required for polycistronic pre-mRNA processing in Caenorhabditis elegans

In Caenorhabditis elegans, polycistronic pre-mRNAs are processed by cleavage and polyadenylation at the 3' ends of the upstream genes and trans splicing, generally to the specialized spliced leader SL2, at the 5' ends of the downstream genes. Previous studies have indicated a relationship between these two events in the processing of a heat shock-induced gpd-2-gpd-3 polycistronic pre-mRNA. Here, we report mutational analysis of the intercistronic region of this operon by linker scan analysis. Surprisingly, no sequences downstream of the 3' end were important for 3'-end formation. In contrast, a U-rich (Ur) element located 29 bp downstream of the site of 3'-end formation was shown to be important for downstream mRNA biosynthesis. This approximately 20-bp element is sufficient for SL2 trans splicing and mRNA accumulation when transplanted to a heterologous context. Furthermore, when the downstream gene was replaced by a gene from another organism, no loss of trans-splicing specificity was observed, suggesting that the Ur element may be the primary signal required for downstream mRNA processing.