The yeast exosome is a complex of 3 3 5 exoribonucleases. Sequence analysis identified putative human homologues for exosome components, although several were found only as expressed sequence tags. Here we report the cloning of full-length cDNAs, which encode putative human homologues of the Rrp40p, Rrp41p, and Rrp46p components of the exosome. Recombinant proteins were expressed and used to raise rabbit antisera. In Western blotting experiments, these decorated HeLa cell proteins of the predicted sizes. All three human proteins were enriched in the HeLa cells nucleus and nucleolus, but were also clearly detected in the cytoplasm. Size exclusion chromatography revealed that hRrp40p, hRrp41p, and hRrp46p were present in a large complex. This cofractionated with the human homologues of other exosome components, hRrp4p and PM/ Scl-100. Anti-PM/Scl-positive patient sera coimmunoprecipitated hRrp40p, hRrp41p, and hRrp46p demonstrating their physical association. The immunoprecipitated complex exhibited 3 3 5 exoribonuclease activity in vitro. hRrp41p was expressed in yeast and shown to suppress the lethality of genetic depletion of yeast Rrp41p. We conclude that hRrp40p, hRrp41p, and hRrp46p represent novel components of the human exosome complex.In both bacteria and eukaryotes, the processing and degradation of many RNA species involves multiprotein complexes (reviewed in Refs. 1-4). The Escherichia coli degradosome includes the endoribonuclease E (RNase E), the 3Ј 3 5Ј exonuclease polynucleotide phosphorylase, the DEAD box RNA helicase RhlB, and several additional proteins whose role is unclear (5-7). Related complexes are implicated in RNA processing in chloroplasts and mitochondria (8 -10). The yeast exosome contains at least 11 components, which are known or predicted to be 3Ј 3 5Ј exoribonucleases (11,12). Ten of these (Rrp4p, Rrp40 -46p, Mtr3p, and Csl4p) have been demonstrated to be encoded by essential genes. These 10 components were found in both cytoplasmic and nuclear complexes, whereas the nonessential RRP6 gene product was detected only in the nucleus (11, 13).The 3Ј processing of many RNAs is affected by the absence or mutation of exosome components. The nuclear exosome is implicated in the processing of ribosomal RNA (rRNA), spliceosomal small nuclear RNAs, and small nucleolar RNAs, as well as the degradation of pre-rRNA spacers and unspliced pre-mRNAs (12-22). The cytoplasmic exosome complex is involved in the 3Ј 3 5Ј pathway of mRNA degradation (22). The activity of the exosome complex may be regulated by cofactors including, for example, the putative ATP-dependent DEVH box RNA helicases Dob1p and Ski2p (23,24).Human cells also contain a multiprotein complex that is related to the yeast exosome (11). This complex, initially designated as the polymyositis/scleroderma (PM/Scl) 1 overlap syndrome particle and herein referred to as the human exosome, was reported to contain 11 (25) to 16 (26) subunits ranging from 20 to 110 kDa. Two proteins of this complex were identified as autoantigens, which are tar...

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 Ul snRNP-specific protein C contains an N-terminal zinc finger-ike CH motif which is required for the binding of the U1C protein to the Ul snRNP particle. Recently a similar motif was reported to be essential for in vivo homodimerization of the yeast splicing factor PRP9. In the present study we demonstrate that the human UIC protein is able to form homodimers as well. Ul C homodimers are found when (i) the human U1C protein is expressed in Escherichia coil, (ii) immunoprecipitations with anti-UI C antibodies are performed on in vitro translated Ul C, and when (iii) the yeast two hybrid system is used. Analyses of mutant Ul C proteins in an in vitro dimerization assay and the yeast two hybrid system revealed that amino acids within the CH motif, i.e. between positions 22 and 30, are required for homodimerization.

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No structural information on U1C protein either in its free state or bound to the spliceosomal U1 small nuclear ribonucleoprotein (snRNP) particle is currently available. Using rabbit antibodies raised against a complete set of 15 U1C overlapping synthetic peptides (16-30 residues long) in different immunochemical tests, linear regions exposed at the surface of free and U1 snRNP-bound U1C were identified. Epitopes within at least three regions spanning residues 31-62, 85-103 and 116-159 were recognized on free and plastic-immobilized recombinant human U1C expressed in Escherichia coli, on in vitro translated U1C protein and on U1C bound to the U1 snRNP particle present in HeLa S100 extract. Using a zinc affinity labeling method, we further showed that the N-terminal U1C peptide containing a zinc-finger motif (peptide 5-34) effectively binds65Zn2+. The N-terminal region of U1C, which is functional in U1 snRNP assembly, is apparently not located at the surface of the U1 snRNP particle.