A bipartite U1 site represses U1A expression by synergizing with PIE to inhibit nuclear polyadenylation

Abstract

U1A protein negatively autoregulates itself by polyadenylation inhibition of its own pre-mRNA by binding as two molecules to a 39UTR-located Polyadenylation Inhibitory Element (PIE). The (U1A)2-PIE complex specifically blocks U1A mRNA biosynthesis by inhibiting polyA tail addition, leading to lower mRNA levels. U1 snRNP bound to a 59ss-like sequence, which we call a U1 site, in the 39UTRs of certain papillomaviruses leads to inhibition of viral late gene expression via a similar mechanism. Although such U1 sites can also be artificially used to potently silence reporter and endogenous genes, no naturally occurring U1 sites have been found in eukaryotic genes. Here we identify a conserved U1 site in the human U1A gene that is, unexpectedly, within a bipartite element where the other part represses the U1 site via a base-pairing mechanism. The bipartite element inhibits U1A expression via a synergistic action with the nearby PIE. Unexpectedly, synergy is not based on stabilizing binding of the inhibitory factors to the 39UTR, but rather is a property of the larger ternary complex. Inhibition targets the biosynthetic step of polyA tail addition rather than altering mRNA stability. This is the first example of a functional U1 site in a cellular gene and of a single gene containing two dissimilar elements that inhibit nuclear polyadenylation. Parallels with other examples where U1 snRNP inhibits expression are discussed. We expect that other cellular genes will harbor functional U1 sites.

U1A Regulates 3′ Processing of the Survival Motor Neuron mRNA

“…Recent work has identified a global role for the U1 snRNP in regulation of alternative polyadenylation (68), and U1 snRNP sites have been shown to work synergistically with direct U1A binding sites on mRNAs (69,70). We did not detect U1 snRNP bound to the SMN 3Ј-UTR either in vitro or in vivo, nor did we identify any U1 snRNP binding sequences in the SMN 3Ј-UTR.…”

The Regulatory Element in the 3′-Untranslated Region of Human Papillomavirus 16 Inhibits Expression by Binding CUG-binding Protein 1

“…Up to now, U1 binding sites had only been found in papillomaviruses and it was of particular interest to determine whether cellular genes utilize U1 binding sites within terminal exons to regulate gene expression. We just recently identified the first example of a mammalian gene having a natural U1 binding site (40). Although this site matches the consensus sequence, and so should be constitutively active, its activity is influenced by two types of flanking sequences, one that represses via an RNA secondary structure and the other that stimulates via binding a trans-acting factor.…”

Analysis of Spliceosomal Proteins in Trypanosomatids Reveals Novel Functions in mRNA Processing

“…The effect of U1A depletion on trans-splicing may stem from its role in polyadenylation, due to the linkage between these processes (38,39). Indeed, U1A was shown to associate with mammalian poly(A) polymerase (44,45). To examine if the effect of U1A depletion on trans-splicing stems from its direct role in polyadenylation, two different approaches were taken; these included purification of protein complexes carrying U1A and examining the direct role of the factor in polyadenylation.…”