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 β1α1β2β3α2β4 structure in which the β strands form a sheet with the highly conserved RNP1 and RNP2 motifs located in the two central β strands, β3 and β1, respectively (14, 23). An additional α helix (helix 3; hereafter referred to as helix C) is present when a longer fragment of the U1A protein is analyzed (aa 2 to 102; reference 15). Using both NMR and X-ray crystallography, the structure of U1A aa 2 to 98 in complex with SL2 of U1RNA has also been solved (1, 2, 15, 24). In this structure, the RNA loop lies across the β sheet, fitting into a groove formed between loop 3 (connecting β2 and β3) and the C-terminal portion of the RRM domain. In spite of intensive investigation, the C-terminal RRM (aa 202 to 283) of U1A does not seem to have any affinity for RNA (21). The U1 snRNP particle is involved in the first step of spliceosome formation, in which it binds to the 5′ splice site of the pre-mRNA (reviewed in reference 18). It is possible that U1A is not essential for the splicing reaction, since in vitro splicing can still proceed in the absence of U1A. It has been suggested, however, that the U1A protein might play an important role in 5′ and 3′ splice site communication (33). In vertebrates, the U1A protein is able to regulate the polyadenylation of U1A pre-mRNA, thereby regulating its own expression level (4). The 3′ untranslated region (UTR) of the human U1A pre-mRNA contains a 50-nucleotide region, designated the polyadenylation-inhibitory element (PIE) RNA, whose sequence and structure are conserved in vertebrates. Located within the PIE RNA are two stretches of seven unpaired nucleotides designated loops 1 and 2, each being able to bind one molecule of U1A protein. Although one of the loops, when studied in isolation, has 27-fold lower affinity for U1A than the other, it was demonstrated that two molecules of U1A bind with high affinity (Kd, ∼0.1 nM) to PIE RNA, indicative of cooperative RNA binding (4, 35). The resulting (U1A)2-PIE RNA complex inhibits addition of the poly(A) tail to the U1A pre-mRNA by specifically inhibiting the enzyme poly(A) polymerase (PAP) (10). Inhibition of polyadenylation requires both the C-terminal 20 residues of PAP and aa 103 to 115 of U1A. A model has been proposed in which the U1A autoregulatory complex inhibits PAP by bringing two copies of U1A aa 103 to 115 into close proximity (11, 34). In support of this model, it was found that two molecules, but not one molecule, of U1A bound to PIE RNA inhibit PAP. Likewise, a monomeric peptide consisting of U1A aa 103 to 115 is unable to inhibit PAP; however, upon increasing its local concentration by conjugation to bovine serum albumin (BSA), this same peptide becomes a potent inhibitor of PAP (11). PAP inhibition by the BSA-peptide conjugate does not require PIE RNA, suggesting that the main role of PIE RNA in PAP inhibition is to bring the two U1A proteins into close proximity. Indeed, the unusual secondary structure of PIE RNA is not essential for inhibition (11, 34). Independent of the biochemical analysis, the determination of the structure by NMR of one molecule of U1A (aa 2 to 98) bound to PIE RNA (1) has also led to the proposal (based on modeling) that the two PIE-RNA-bound U1A proteins make extensive protein-protein interactions throughout the N-terminal 100 residues (16). Here we show, by using both the yeast two-hybrid system and in vitro assays, that U1A is able to homodimerize in the absence of RNA sequences that specifically bind U1A (i.e., SL2 of U1RNA and PIE RNA). Dimerization requires two regions, both located in the N-terminal 115 residues. Mutations of the second region (aa 103 to 115) which abolish dimerization also result in either reduction or complete loss of cooperative binding to PIE RNA but with no effect on U1A binding as a monomer to PIE RNA. These same mutations also result in loss of U1A's ability to inhibit polyadenylation. A dimeric form of a peptide containing these residues also inhibits polyadenylation. A model integrating these results will be presented explaining how the U1A autoregulatory complex functions.