Determinants within an 18-Amino-Acid U1A Autoregulatory Domain That Uncouple Cooperative RNA Binding, Inhibition of Polyadenylation, and Homodimerization

Abstract

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...

Regulation of Yeast NRD1 Expression by Premature Transcription Termination

“…Subsequent phosphorylation of the Pol II CTD and negative elongation factor DSIF/NELF leads to readthrough of this terminator (reviewed in Garriga and Grana [2004], Price [2000]). No equivalent eukaryotic cellular regulatory system has previously been described, although several examples of RNA binding proteins directing alternative 3 0 end formation have been reported (Guan et al, 2003;Roth et al, 2005).…”

Role of Cajal Bodies and Nucleolus in the Maturation of the U1 snRNP in Arabidopsis

“…Interestingly, the yeast U1C protein was found to bind to the 5′ splice site in the absence of pre-mRNA-U1 snRNP base pairing [49] . In human cells the U1A protein, aside from its role in snRNP function, exists in a snRNP-free fraction which is involved in regulation of its own expression level and in regulation of polyadenylation of various cellular pre-mRNAs [50] , [51 and references therein] . Hence, it is well possible that the predominant diffuse nucleoplasmic localisation of Arabidopsis U1A and U1C proteins also reflects their additional functions, apart from U1 snRNP and pre-mRNA splicing.…”

Differential expression of eIF5A‐1 and eIF5A‐2 in human cancer cells

“…Regulated polyadenylation depends on the interaction of RNA‐binding proteins at specific sites close to the cleavage site in the 3′‐UTR of the pre‐mRNA. The best example comes from the snRNP protein U1A which autoregulates its polyadenylation by binding to its pre‐mRNA and directly interacts with and inhibits the poly(A) polymerase [41–43]. The second example involves the switch from cell‐attached to secreted IgM by B‐cells [44,45].…”