A set of seven Sm proteins assemble on the Sm-binding site of spliceosomal U snRNAs to form the ring-shaped Sm core. The U7 snRNP involved in histone RNA 3 processing contains a structurally similar but biochemically unique Sm core in which two of these proteins, Sm D1 and D2, are replaced by Lsm10 and by another as yet unknown component. Here we characterize this factor, termed Lsm11, as a novel Sm-like protein with apparently two distinct functions. In vitro studies suggest that its long N-terminal part mediates an important step in histone mRNA 3-end cleavage, most likely by recruiting a zinc finger protein previously identified as a processing factor. In contrast, the C-terminal part, which comprises two Sm motifs interrupted by an unusually long spacer, is sufficient to assemble with U7, but not U1, snRNA. Assembly of this U7-specific Sm core depends on the noncanonical Sm-binding site of U7 snRNA. Moreover, it is facilitated by a specialized SMN complex that contains Lsm10 and Lsm11 but lacks Sm D1/D2. Thus, the U7-specific Lsm11 protein not only specifies the assembly of the U7 Sm core but also fulfills an important role in U7 snRNP-mediated histone mRNA processing.[Keywords: Small nuclear ribonucleoprotein; histone pre-mRNA 3Ј processing; Sm-like protein; Sm core structure; SMN-Gemin complex] Sm and Sm-like (Lsm) proteins have been found in eukaryotes, archaea, and eubacteria. They are characterized by two closely spaced, conserved Sm motifs 1 and 2 (Hermann et al. 1995;Seraphin 1995) which adopt a fold consisting of an ␣-helix followed by five -strands (Kambach et al. 1999b). A common characteristic of Sm/Lsm proteins is their tendency to form oligomers that can close into hepta-or hexameric ring structures which, in turn, control various aspects of RNA metabolism.The seven prototype Sm proteins B/BЈ, D1, D2, D3, E, F, and G form the so-called Sm core structure around the conserved Sm-binding site, RAUU U / G UUGR, of the spliceosomal small nuclear RNAs (snRNAs; Lührmann et al. 1990;Raker et al. 1996;Kambach et al. 1999a;). The formation of this structure occurs in the cytoplasm and was recently shown to be ATP-dependent and mediated by specific assembly factors. A key player in this process is the "survival of motor neurons" (SMN) protein which is mutated in the neuromuscular disorder spinal muscular atrophy (for review, see Paushkin et al. 2002). SMN is part of the so-called SMN complex, which is composed of at least 18 distinct proteins, including all Sm proteins. In vitro reconstitution of the assembly reaction revealed that Sm proteins first associate with the SMN complex and are subsequently transferred to the U snRNA (Meister et al. 2001a;Pellizzoni et al. 2002). The functioning of the SMN complex is regulated by the PRMT5 complex. This complex introduces symmetrical dimethylarginines in Sm proteins B/BЈ, D1, and D3, thereby increasing their affinity for SMN (Brahms et al. 2001;Friesen et al. 2001;Meister et al. 2001b).In contrast to the canonical Sm proteins found in the Sm core of spliceosomal...
U7 snRNPs were isolated from HeLa cells by biochemical fractionation, followed by affinity purification with a biotinylated oligonucleotide complementary to U7 snRNA. Purified U7 snRNPs lack the Sm proteins D1 and D2, but contain additional polypeptides of 14, 50 and 70 kDa. Microsequencing identified the 14 kDa polypeptide as a new Sm-like protein related to Sm D1 and D3. Like U7 snRNA, this protein, named Lsm10, is enriched in Cajal bodies of the cell nucleus. Its incorporation into U7 snRNPs is largely dictated by the special Sm binding site of U7 snRNA. This novel type of Sm complex, composed of both conventional Sm proteins and the Sm-like Lsm10, is most likely to be important for U7 snRNP function and subcellular localization.
The hairpin structure at the 3' end of animal histone mRNAs controls histone RNA 3' processing, nucleocytoplasmic transport, translation and stability of histone mRNA. Functionally overlapping, if not identical, proteins binding to the histone RNA hairpin have been identified in nuclear and polysomal extracts. Our own results indicated that these hairpin binding proteins (HBPs) bind their target RNA as monomers and that the resulting ribonucleoprotein complexes are extremely stable. These features prompted us to select for HBP-encoding human cDNAs by RNA-mediated three-hybrid selection in Saccharomyces cerevesiae. Whole cell extract from one selected clone contained a Gal4 fusion protein that interacted with histone hairpin RNA in a sequence- and structure-specific manner similar to a fraction enriched for bovine HBP, indicating that the cDNA encoded HBP. DNA sequence analysis revealed that the coding sequence did not contain any known RNA binding motifs. The HBP gene is composed of eight exons covering 19.5 kb on the short arm of chromosome 4. Translation of the HBP open reading frame in vitro produced a 43 kDa protein with RNA binding specificity identical to murine or bovine HBP. In addition, recombinant HBP expressed in S. cerevisiae was functional in histone pre-mRNA processing, confirming that we have indeed identified the human HBP gene.
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