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A Minimal SMN Complex Facilitates Formation of Usnrnps in Drosophila Melanogaster

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A Minimal SMN Complex Facilitates Formation of Usnrnps in Drosophila Melanogaster Evolution of an RNP assembly system: A minimal SMN complex facilitates formation of UsnRNPs in Drosophila melanogaster Matthias Kroiss*, Jo¨ rg Schultz†, Julia Wiesner‡, Ashwin Chari*, Albert Sickmann‡, and Utz Fischer*§ Departments of *Biochemistry and †Bioinformatics, Theodor Boveri Institute, Biocenter, Am Hubland, 97074 Wu¨rzburg, Germany; and ‡Functional Genomics Group, Rudolf Virchow Center, Versbacher Strasse 9, 97078 Wu¨rzburg, Germany Edited by Kay E. Davies, University of Oxford, Oxford, United Kingdom, and accepted by the Editorial Board April 25, 2008 (received for review March 8, 2008) In vertebrates, assembly of spliceosomal uridine-rich small nuclear The SMN complex not only functions in the assembly of the ribonucleoproteins (UsnRNPs) is mediated by the SMN complex, a Sm core domain, but also influences additional steps in the macromolecular entity composed of the proteins SMN and Gemins biogenesis pathway of UsnRNPs. One such step is the nuclear 2–8. Here we have studied the evolution of this machinery using import of the assembled UsnRNP, which is mediated by the complete genome assemblies of multiple model organisms. The SMN complex (or parts thereof) in conjunction with the import SMN complex has gained complexity in evolution by a blockwise factor importin ␤ (9). In addition, specific UsnRNP proteins addition of Gemins onto an ancestral core complex composed of and the cap hypermethylase Tgs1 have been found in associa- SMN and Gemin2. In contrast to this overall evolutionary trend to tion of the SMN complex (10). This observation indicates that more complexity in metazoans, orthologs of most Gemins are the SMN complex coordinates various events during UsnRNP missing in dipterans. In accordance with these bioinformatic data biogenesis by assuming the role of a binding platform for the a previously undescribed biochemical purification strategy eluci- respective assisting factors. dated that the dipteran Drosophila melanogaster contains an SMN The multisubunit composition of the human SMN complex complex of remarkable simplicity. Surprisingly, this minimal com- has impeded the mechanistic dissection of the UsnRNP assembly plex not only mediates the assembly reaction in a manner very process. Thus, although RNA interference studies indicated similar to its vertebrate counterpart, but also prevents misassem- essential roles of several Gemins in the assembly reaction bly onto nontarget RNAs. Our data suggest that only a minority of (11–13), their precise contributions remain unclear. To facilitate Gemins are required for the assembly reaction per se, whereas mechanistic studies and to gain insight into the evolution of the others may serve additional functions in the context of UsnRNP SMN complex, we have mined genomic databases for organisms biogenesis. The evolution of the SMN complex is an interesting that lack individual Gemins and hence may contain a simpler example of how the simplification of a biochemical process con- assembly machinery. Indeed, this was the case for different tributes to genome compaction. organisms including dipterans. We chose to further investigate Drosophila because of the wealth of genetic resources. An splicing ͉ spinal muscular atrophy ͉ UsnRNA affinity chromatography strategy has permitted us the purifica- tion of an assembly-active complex composed of SMN and Gemin2 only. Remarkably, this complex not only facilitated plicing of pre-mRNAs is catalyzed by the spliceosome, a assembly of the Sm core domain but also discriminated between Smacromolecular machine consisting of a large number of cognate and noncognate RNAs. Thus, our combined bioinfor- protein factors and the uridine-rich small nuclear ribonucleo- matic and biochemical approach revealed that the assembly EVOLUTION proteins (snRNPs) U1, U2, U4/6, and U5. The biogenesis of reaction requires only two core proteins in vitro, even though these particles occurs in a stepwise manner. First, nuclear- SMN complexes from most metazoans are of considerable 7 transcribed, m G-capped snRNAs U1, U2, U4, and U5 are complexity. We speculate that Gemins 3–8 have been recruited exported into the cytoplasm, where a conserved sequence motif to the SMN complex in the course of evolution to integrate in these RNAs (Sm site) serves as a binding platform for the assembly with additional steps in the biogenesis of UsnRNPs. seven Sm proteins B/BЈ, D1, D2, D3, E, F, and G. As a consequence, a ring-shaped Sm core domain is formed. This Results domain is crucial for subsequent steps in the biogenesis of An Elaborate SMN Complex Is Characteristic of Metazoans. To un- UsnRNPs, such as formation of the hypermethylated m2,2,7G cap derstand how the UsnRNP assembly machinery has evolved, we and import of the assembled particle into the nucleus. At a yet performed homology searches for all Gemin proteins constituting to be defined step, additional factors are recruited to form the the human SMN complex in genomic databases of a variety of mature UsnRNP particles that function in splicing (1). organisms (see Experimental Procedures for details). Because of its Previous studies have shown that Sm proteins bind spontane- diverse functions (14, 15) and its transient cytoplasmic interaction ously, albeit in a hierarchical manner, onto UsnRNAs in vitro (2, 3). However, in cellular extracts, this process depends on ATP and the activity of the multisubunit SMN complex (4–6). Author contributions: M.K., J.S., A.C., and U.F. designed research; M.K., J.S., J.W., and A.C. performed research; M.K. contributed new reagents/analytic tools; M.K., J.S., J.W., A.S., and Recently, a systematic interaction study on the human SMN U.F. analyzed data; and M.K., J.S., A.C., and U.F. wrote the paper. complex has established its basic architecture (7). A modular Conflict of interest statement: The authors have filed a patent application for the affinity composition was deduced where the three factors SMN, Gemin2, purification of complexes (European Patent Office application no. 07108779.5). and Gemin8 form the backbone of the entire complex. Onto this This article is a PNAS Direct Submission. K.E.D. is a guest editor invited by the Editorial Board. core, the peripheral building blocks Gemin3/4 and Gemin6/7/ §To whom correspondence should be addressed. E-mail: utz.fischer@biozentrum. UNRIP bind to form the functional unit. In support of this uni-wuerzburg.de. modular architecture, Gemin-containing subcomplexes have This article contains supporting information online at www.pnas.org/cgi/content/full/ been identified composed of SMN/Gemin2, Gemin3–Gemin5, 0802287105/DCSupplemental. and Gemin6/7/UNRIP (8). © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0802287105 PNAS ͉ July 22, 2008 ͉ vol. 105 ͉ no. 29 ͉ 10045–10050 Downloaded by guest on September 24, 2021 Fig. 1. Evolution of the SMN complex. Complete ge- nome assemblies of indicated organisms have been screened for orthologs of human SMN complex compo- nents. The presence of the proteins was mapped on a published phylogenetic tree (38). A homolog of Gemin6 was found in the algae O. tauri, but the homology was restricted to the C-terminus and therefore is not shown. Gemin5 orthologs in dipterans are evolving significantly faster than in other organisms. Because this may indicate a change of function, they are shown as squares. with the SMN complex, the UNRIP protein has been excluded Gemin4 first appeared in the sea urchin Strongylocentrotus purpu- from this analysis. ratus, whereas it is absent in all ecdysozoans under study. This SMN and Gemin2 orthologs (termed Yab8p and Yip1p) but no suggests that Gemin4 has joined the SMN complex only recently in other Gemins can be found in the fungus Schizosaccharomyces evolution, most likely with the appearance of deuterostomians. pombe [see Fig. 1; for protein identifiers see supporting information Consequently, it was found to be part of the SMN complex in (SI) Table S1]. Importantly, both orthologs interact physically and vertebrates such as Danio rerio but also cephalochordates like may hence form a functional unit (16). In Saccharomyces cerevisiae, Branchiostoma floridae and Ciona intestinalis (urochordates). Thus, however, only the distantly related Gemin2 ortholog Brr1p, but no plants and some fungi possess a core complex composed of SMN SMN ortholog, could be identified. Brr1p has been proposed to be and Gemin2 only, whereas an elaborate SMN complex has devel- an ortholog of human Gemin2 in an early publication (5). However, oped only in animal branches by addition of Gemin proteins. because of its limited homology to Yip1p this finding has been questioned (16). Taking advantage of the dramatically increased Absence of Most Gemin Orthologs in Genomes of Dipterans. The genome databases and novel search algorithms (PSI BLAST), bioinformatic data indicated an evolutionary trend in the animal Brr1p can be defined as the single significant homolog of human kingdom toward a multisubunit SMN complex. Interestingly, how- Ϫ ever, we failed to identify orthologs of most Gemins in the dipterans Gemin2 (NP࿝003607.1, PSI BLAST third iteration, E ϭ 3 ϫ 10 5) Ϫ Drosophila melanogaster and Anopheles gambiae although they were and S. pombe Yip1p (NP࿝594775.1, fourth iteration, E ϭ 3 ϫ 10 5). present in closely related Apis mellifera and Nasonia vitripennis.We Because reciprocal searches further support this homology, Brr1p have restricted our further analysis to D. melanogaster in this study. is the ortholog of human Gemin2. Thus, S. cerevisiae retained only Besides the known SMN ortholog (17, 18), we found a Gemin2 one Gemin and hence is unlikely to form a functional SMN ortholog encoded by CG10419 and putative orthologs of Gemin3 complex. Interestingly, like S. pombe, the plants Arabidopsis thaliana (Dhh1) and Gemin5 (Rigor mortis). Dhh1 protein shows high and Oryza sativa contained orthologs of SMN and Gemin2 only. We conservation in the N-terminal DEAD box helicase domain but therefore conclude that SMN and Gemin2 represent the most possesses a diverged C terminus.
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