The prothrombin (F2) 3' end formation signal is highly susceptible to thrombophilia-associated gain-of-function mutations. In its unusual architecture, the F2 3' UTR contains an upstream sequence element (USE) that compensates for weak activities of the non-canonical cleavage site and the downstream U-rich element. Here, we address the mechanism of USE function. We show that the F2 USE contains a highly conserved nonameric core sequence, which promotes 3' end formation in a position- and sequence-dependent manner. We identify proteins that specifically interact with the USE, and demonstrate their function as trans-acting factors that promote 3' end formation. Interestingly, these include the splicing factors U2AF35, U2AF65 and hnRNPI. We show that these splicing factors not only modulate 3' end formation via the USEs contained in the F2 and the complement C2 mRNAs, but also in the biocomputationally identified BCL2L2, IVNS and ACTR mRNAs, suggesting a broader functional role. These data uncover a novel mechanism that functionally links the splicing and 3' end formation machineries of multiple cellular mRNAs in an USE-dependent manner.

The nonsense-mediated mRNA decay (NMD) pathway promotes the rapid degradation of mRNAs containing premature stop codons (PTCs). In Caenorhabditis elegans, seven genes (smg1-7) playing an essential role in NMD have been identified. Only SMG2-4 (known as UPF1-3) have orthologs in Saccharomyces cerevisiae. Here we show that the Drosophila orthologs of UPF1-3, SMG1, SMG5 and SMG6 are required for the degradation of PTC-containing mRNAs, but that there is no SMG7 ortholog in this organism. In contrast, orthologs of SMG5-7 are encoded by the human genome and all three are required for NMD. In human cells, exon boundaries have been shown to play a critical role in defining PTCs. This role is mediated by components of the exon junction complex (EJC). Contrary to expectation, however, we show that the components of the EJC are dispensable for NMD in Drosophila cells. Consistently, PTC definition occurs independently of exon boundaries in DROSOPHILA: Our findings reveal that despite conservation of the NMD machinery, different mechanisms have evolved to discriminate premature from natural stop codons in metazoa.

One year after the release of the sequence of yeast chromosome III, we have re-examined its open reading frames (ORFs) by computer methods. More than 61% of the 171 probable gene products have significant sequence similarities in the current databases; as many as 54% have already known functions or are related to functionally characterized proteins, allowing partial prediction of protein function, 11 percentage points more than reported a year ago; 19% are similar to proteins of known three-dimensional structure, allowing model building by homology. The most interesting new identifications include a sugar kinase distantly related to ribokinases, a phosphatidyl serine synthetase, a putative transcription regulator, a flavodoxin-like protein, and a zinc finger protein belonging to a distinct subfamily. Several ORFs have similarities to uncharacterized proteins, resulting in new families in search of a function'. About 54% of ORFs match sequences from other phyla, including numerous fragments in the database of expressed sequence tags (ESTs). Most significant similarities to ESTs are with proteins in conserved families widely represented in the databases. About 30% of ORFs contain one or more predicted transmembrane segments. The increase in the power of functional and structural prediction comes from improvements in sequence analysis and from richer databases and is expected to facilitate substantially the experimental effort in characterizing the function of new gene products.