The regulation of polyadenylation by U1A protein and U1snRNP

The 3'-end processing of nearly all eukaryotic pre-mRNAs comprises the cleavage and polyadenylation steps. The 3'-polyA tail is essential for translation, mRNA stability and export, thus poly(A) regulation is important for these functions. The human U1A protein autoregulates its own production by inhibiting the polyadenylation of its own pre-mRNA via binding to a PIE site. Residues 103 to 115 are essential for autoregulation and contain 3 distinct activities: cooperative binding of two U1As to the PIE site, formation of a novel homodimerization surface, and inhibition of polyadenylation. Here we purified and analyzed 11 substitution mutants in this region. In 5 mutants we found particular amino acids associate with one activity but not another indicating they can be uncoupled. Surprisingly, for 3 mutants these activities are improved suggesting U1A autoregulation is selected for sub-optimal inhibitory efficiency. The effect of these mutations on autoregulatory activity in vivo was also determined.; U1 snRNA can specifically inhibit reporter gene expression by inhibiting polyadenylation when its 5'-end basepairs to a so-called U1 site near the poly(A) signal of the reporter gene's pre-mRNA. Here we demonstrate that expression of endogenous mammalian genes can be efficiently inhibited by transiently-expressed 5'-end-mutated U1 snRNA. Also, we determined the inhibitory mechanism and established a set of rules to use and improve this technique. Two U1 snRNAs basepaired to two U1 sites on a single pre-mRNA act synergistically. The U1 snRNA must be targeted to the terminal exon of the pre-mRNA and it only inhibits the polyadenylation of a downstream poly(A) signal.; Putative U1 sites are found in many cellular genes' 3'-UTRs. Here we report that a putative U1 site in the U1A 3'-UTR and its distance to the PIE site are conserved among mammals, implying functional significance. Investigation in the context of a reporter gene indicated similar and independent inhibitory activity of both sites, whereas in the context of expression of the U1A cDNA, only the PIE site showed a significant inhibitory activity. However, when both were present there was a boost of inhibition indicating a synergistic effect between the two sites. In vitro assays were employed to determine the mechanism of this synergism. A model involving a repressor of this U1 site was proposed.