Mutational analysis of 3' splice site selection during trans-splicing

trans-Splicing is essential for mRNA maturation in trypanosomatids. A conserved AG dinucleotide serves as the 3' splice acceptor site, and analysis of native processing sites suggests that selection of this site is determined according to a 5'-3' scanning model. A series of stable gene replacement lines were generated that carried point mutations at or near the 3' splice site within the intergenic region separating CUB2.65, the calmodulin-ubiquitin associated gene, and FUS1, the ubiquitin fusion gene of Trypanosoma cruzi. In one stable line, the elimination of the native 3' splice acceptor site led to the accumulation of Y-branched splicing intermediates, which served as templates for mapping the first trans-splicing branch points in T. cruzi. In other lines, point mutations shifted the position of the first consensus AG dinucleotide either upstream or downstream of the wild-type 3' splice acceptor site in this intergenic region. Consistent with the scanning model, the first AG dinucleotide downstream of the branch points was used as the predominant 3' splice acceptor site. In all of the stable lines, the point mutations affected splicing efficiency in this region.     

A 57-nucleotide upstream early polyadenylation element in human papillomavirus type 16 interacts with hFip1, CstF-64, hnRNP C1/C2, and polypyrimidine tract binding protein

We have investigated the role of the human papillomavirus type 16 (HPV-16) early untranslated region (3' UTR) in HPV-16 gene expression. We found that deletion of the early 3' UTR reduced the utilization of the early polyadenylation signal and, as a consequence, resulted in read-through into the late region and production of late L1 and L2 mRNAs. Deletion of the U-rich 3' half of the early 3' UTR had a similar effect, demonstrating that the 57-nucleotide U-rich region acted as an enhancing upstream element on the early polyadenylation signal. In accordance with this, the newly identified hFip1 protein, which has been shown to enhance polyadenylation through U-rich upstream elements, interacted specifically with the HPV-16 upstream element. This upstream element also interacted specifically with CstF-64, hnRNP C1/C2, and polypyrimidine tract binding protein, suggesting that these factors were either enhancing or regulating polyadenylation at the HPV-16 early polyadenylation signal. Mutational inactivation of the early polyadenylation signal also resulted in increased late mRNA production. However, the effect was reduced by the activation of upstream cryptic polyadenylation signals, demonstrating the presence of additional strong RNA elements downstream of the early polyadenylation signal that direct cleavage and polyadenylation to this region of the HPV-16 genome. In addition, we identified a 3' splice site at genomic position 742 in the early region with the potential to produce E1 and E4 mRNAs on which the E1 and E4 open reading frames are preceded only by the suboptimal E6 AUG. These mRNAs would therefore be more efficiently translated into E1 and E4 than previously described HPV-16 E1 and E4 mRNAs on which E1 and E4 are preceded by both E6 and E7 AUGs.     

Aberrant mRNAs with extended 3' UTRs are substrates for rapid degradation by mRNA surveillance

The mRNA surveillance system is known to rapidly degrade aberrant mRNAs that contain premature termination codons in a process referred to as nonsense-mediated decay. A second class of aberrant mRNAs are those wherein the 3' UTR is abnormally extended due to a mutation in the polyadenylation site. We provide several observations that these abnormally 3'-extended mRNAs are degraded by the same machinery that degrades mRNAs with premature nonsense codons. First, the decay of the 3'-extended mRNAs is dependent on the same decapping enzyme and 5'-to-3' exonuclease. Second, the decay is also dependent on the proteins encoded by the UPF1, UPF2, and UPF3 genes, which are known to be specifically required for the rapid decay of mRNAs containing nonsense codons. Third, the ability of an extended 3' UTR to trigger decay is prevented by stabilizing sequences within the PGK1 coding region that are known to protect mRNAs from the rapid decay induced by premature nonsense codons. These results indicate that the mRNA surveillance system plays a role in degrading abnormally extended 3' UTRs. Based on these results, we propose a model in which the mRNA surveillance machinery degrades aberrant mRNAs due to the absence of the proper spatial arrangement of the translation-termination codon with respect to the 3' UTR element as defined by the utilization of a polyadenylation site.