Fourteen Residues of the U1 snRNP-Specific U1A Protein Are Required for Homodimerization, Cooperative RNA Binding, and Inhibition of Polyadenylation

Abstract

It was previously shown that the human U1A protein, one of three U1 small nuclear ribonucleoproteinspecific proteins, autoregulates its own production by binding to and inhibiting the polyadenylation of its own pre-mRNA. The U1A autoregulatory complex requires two molecules of U1A protein to cooperatively bind a 50-nucleotide polyadenylation-inhibitory element (PIE) RNA located in the U1A 3 untranslated region. Based on both biochemical and nuclear magnetic resonance structural data, it was predicted that protein-protein interactions between the N-terminal regions (amino acids [aa] 1 to 115) of the two U1A proteins would form the basis for cooperative binding to PIE RNA and for inhibition of polyadenylation. In this study, we not only experimentally confirmed these predictions but discovered some unexpected features of how the U1A autoregulatory complex functions. We found that the U1A protein homodimerizes in the yeast two-hybrid system even when its ability to bind RNA is incapacitated. U1A dimerization requires two separate regions, both located in the N-terminal 115 residues. Using both coselection and gel mobility shift assays, U1A dimerization was also observed in vitro and found to depend on the same two regions that were found in vivo. Mutation of the second homodimerization region (aa 103 to 115) also resulted in loss of inhibition of polyadenylation and loss of cooperative binding of two U1A protein molecules to PIE RNA. This same mutation had no effect on the binding of one U1A protein molecule to PIE RNA. A peptide containing two copies of aa 103 to 115 is a potent inhibitor of polyadenylation. Based on these data, a model of the U1A autoregulatory complex is presented.The U1 small nuclear ribonucleoprotein (snRNP) particle is the most abundant member of the spliceosomal snRNPs. Human U1 snRNP is comprised of 10 proteins and the 164-nucleotide U1 small nuclear RNA (U1RNA) and is required for splicing of pre-mRNA (38). One of the U1 snRNP-specific proteins, the U1A protein, contains two evolutionarily conserved RNA recognition motifs (RRMs) characteristic of a large family of proteins involved in the biosynthesis of cellular RNA (reviewed in reference 37). The signature motifs for the RRM family consist of two ribonucleoprotein (RNP) sequences, RNP1 and RNP2, which are the most conserved features of this family. The N-terminal RRM of U1A is, together with some flanking amino acids, necessary and sufficient for binding to the loop part of stem-loop 2 (SL2) sequence AUUGCAC of U1RNA (22,27,28). The structure of the N-terminal RRM of the U1A protein (amino acids [aa] 2 to 95) has been solved both by X-ray crystallography and by nuclear magnetic resonance (NMR) and consists of a ␤ 1 ␣ 1 ␤ 2 ␤ 3 ␣ 2 ␤ 4 structure in which the ␤ strands form a sheet with the highly conserved RNP1 and RNP2 motifs located in the two central ␤ strands, ␤ 3 and ␤ 1 , respectively (14, 23). An additional ␣ helix (helix 3; hereafter referred to as helix C) is present when a longer fragment of the U1A protein is analyzed (aa 2 to ...

Sequences Adjacent to the 5′ Splice Site Control U1A Binding Upstream of the IgM Heavy Chain Secretory Poly(A) Site

“…In this way they form a bi-partite binding pocket for PAP that is necessary to inhibit its activity (14,25,37). We show here that U1A behaves in the same way in that failure to bind a second U1A molecule abolishes the ability of U1A to inhibit PAP activity at the secretory poly(A) site.…”

Oligomerization of EDEN‐BP is required for specific mRNA deadenylation and binding

“…Upon RNA binding, AUF1 dimers are sequentially recruited to the host RNA, forming tetramers and higher polymers of dimers (Wilson et al, 1999). U1A is able to homodimerize, even in mutants that have lost their capacity to interact with RNA; however, in the wildtype protein, homodimerization permits co-operative binding (Klein Gunnewiek et al, 2000). A mutant FMR1 (Fragile X mental retardation 1) I304N that is unable to oligomerize can still interact with RNA (Laggerbauer et al, 2001).…”

TcUBP-1, an mRNA Destabilizing Factor from Trypanosomes, Homodimerizes and Interacts with Novel AU-rich Element- and Poly(A)-binding Proteins Forming a Ribonucleoprotein Complex

“…Both RRM1 and RRM2 are important for ARE affinity, even though they are not sufficient (24). It is important to conclude that certain RNA-binding proteins require only one domain to achieve RNA-binding specificity and affinity, such as U1A (28). Conversely, other proteins have been shown to require combinations of at least two (27) or occasionally more domains (29).…”