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 ...
The human U1 snRNP-specific U1A protein autoregulates its own production by binding to and inhibiting the polyadenylation of its own pre-mRNA. Previous work demonstrated that a short sequence of U1A protein is essential for autoregulation and contains three distinct activities, which are (i) cooperative binding of two U1A proteins to a 50-nucleotide region of U1A pre-mRNA called polyadenylation-inhibitory element RNA, (ii) formation of a novel homodimerization surface, and (iii) inhibition of polyadenylation by inhibition of poly(A) polymerase (PAP). In this study, we purified and analyzed 11 substitution mutant proteins, each having one or two residues in this region mutated. In 5 of the 11 mutant proteins, we found that particular amino acids associate with one activity but not another, indicating that they can be uncoupled. Surprisingly, in three mutant proteins, these activities were improved upon, suggesting that U1A autoregulation is selected for suboptimal inhibitory efficiency. The effects of these mutations on autoregulatory activity in vivo were also determined. Only U1A and U170K are known to regulate nuclear polyadenylation by PAP inhibition; thus, these results will aid in determining how widespread this type of regulation is. Our molecular dissection of the consequences of conformational changes within an RNP complex presents a powerful example to those studying more complicated pre-mRNA-regulatory systems.The U1 small nuclear ribonucleoprotein (snRNP) is the most abundant member of the spliceosomal snRNPs in vertebrate cells. Human U1 snRNP is required for splicing of pre-mRNA and is composed of the 164-nucleotide (nt) U1 small nuclear RNA (snRNA) and 10 polypeptides, 3 of which are specific to U1 snRNP (34). One of these U1 snRNPspecific proteins, U1A, contains two conserved RNA recognition motifs (RRMs) characteristic of the largest family of RNA binding proteins (reviewed in references 3, 25, and 31). Independent of the other U1 snRNP proteins, the N-terminal 101 residues of U1A (U1A 1-101 ), containing one of these RRMs, is sufficient to bind to stem-loop 2 (SL2) of U1 snRNA (22,27) and the U1A-SL2 complex has been the subject of intense biochemical and structural studies. Indeed, of the more than 1,000 RRMs known, the N-terminal RRM of U1A is the best understood at the biochemical and structural levels. RRMs are about 80 amino acids in length and consist of a  1 ␣ 1  2  3 ␣ 2  4 structure in which the four  strands form a sheet buttressed by two ␣ helices (13, 23; see Fig. 1A). Usually, the RRM is sufficient for RNA binding activity; however, in the case of U1A, additional flanking sequences in the form of a third ␣ helix, helix C (residues 92 to 98), are necessary (1,7,11,14,15,19). In stark contrast to the N-terminal RRM, the C-terminal RRM of U1A has low affinity for RNA and no cellular RNA targets have been identified (21).U1 snRNP is involved in early steps of spliceosome formation and binds to the 5Ј splice site of the pre-mRNA (reviewed in reference 18). The function of U1 snRNP-b...
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