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Direct Probing of RNA Structure and RNA-Protein Interactions in Purified Hela Cell&Apos;S and Yeast Spliceosomal U4/U6.U5

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Direct Probing of RNA Structure and RNA-Protein Interactions in Purified Hela Cell&Apos;S and Yeast Spliceosomal U4/U6.U5 doi:10.1006/jmbi.2002.5451availableonlineathttp://www.idealibrary.comon J. Mol. Biol. (2002) 317, 631±649 Direct Probing of RNA Structure and RNA-Protein Interactions in Purified HeLa Cell's and Yeast Spliceosomal U4/U6.U5 Tri-snRNP Particles AnnieMougin1,AlexanderGottschalk2,PatriziaFabrizio2 ReinhardLuÈhrmann2andChristianeBranlant1* 1UMR 7567 CNRS-UHP The U4/U6.U5 tri-snRNP is a key component of spliceosomes. By using Nancy I, Maturation des ARN chemical reagents and RNases, we performed the ®rst extensive exper- et Enzymologie MoleÂculaire imental analysis of the structure and accessibility of U4 and U6 snRNAs Universite H. Poincare in tri-snRNPs. These were puri®ed from HeLa cell nuclear extract and B.P. 239, 54506 Vandoeuvre- Saccharomyces cerevisiae cellular extract. U5 accessibility was also investi- les Nancy CeÂdex, France gated. For both species, data demonstrate the formation of the U4/U6 Y- shaped structure. In the human tri-snRNP and U4/U6 snRNP, U6 forms 2Department of Cellular the long range interaction, that was previously proposed to be respon- Biochemistry, Max-Planck- sible for dissociation of the deproteinized U4/U6 duplex. In both yeast Institute of Biophysical and human tri-snRNPs, U5 is more protected than U4 and U6, Chemistry, D-37077, GoÈttingen suggesting that the U5 snRNP-speci®c protein complex and other com- Germany ponents of the tri-snRNP wrapped the 50 stem-loop of U5. Loop I of U5 is partially accessible, and chemical modi®cations of loop I were identical in yeast and human tri-snRNPs. This re¯ects a strong conservation of the interactions of proteins with the functional loop I. Only some parts of the U4/U6 Y-shaped motif (the 50 stem-loop of U4 and helix II) are pro- tected. Due to difference of protein composition of yeast and human tri- snRNP, the U6 segment linking the 50 stem-loop to the Y-shaped struc- ture and the U4 central single-stranded segment are more accessible in the yeast than in the human tri-snRNP, especially, the phylogenetically conserved ACAGAG sequence of U6. Data are discussed taking into account knowledge on RNA and protein components of yeast and human snRNPs and their involvement in splicesome assembly. # 2002 Elsevier Science Ltd. Keywords: yeast; HeLa cells; splicing; U4/U6.U5 tri-snRNP; RNA *Corresponding author structure and accessibility Introduction lar elements (snRNPs U1, U2 and U4/U6.U5) and numerousnon-snRNPproteins.1±4Whereas,the Spliceosomes that catalyze intron removal in U1 and U2 snRNPs are associated as individual pre-messenger RNAs are formed by sequential particles, the U4/U6 snRNP interacts with the U5 assembly on selected pre-mRNA regions of modu- snRNP and additional proteins to form a stable 25 S tri-snRNP (U4/U6.U5 tri-snRNP), which is integrated in the pre-spliceosomal complex. Gen- Present addresses: A. Mougin, Laboratoire de Biologie MoleÂculaire Eucaryote du CNRS, Universite Paul etic and phylogenetic evidences revealed that U4 Sabatier, 31062 Toulouse, France; A. Gottschalk, and U6 snRNAs interact by formation of two inter- University of California, San Diego, Department of molecularhelicesintheU4/U6snRNP.5However, Biology, 4406 Bonner Hall, 9500 Gilman Drive, La Jolla, up to now, no direct experimental study of the CA 92093-0349, U.S.A. resulting Y-shaped RNA duplex has been per- Abbreviations used: snRNA, small nuclear RNA; formed. RNP, ribonuclear protein; DMS, dimethyl sulfate; Based largely on analysis of splicing complexes CMCT, cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate. in non-denaturing gels, a sequential order of E-mail address of the corresponding author: addition of the snRNPs has been de®ned: ®rst, rec- [email protected] ognition of the 50 splice junction by the U1 snRNP, 0022-2836/02/050631±19 $35.00/0 # 2002 Elsevier Science Ltd. 632 RNA Probing in Yeast and Human U4/U6.U5 Tri-snRNPs then interaction of the branch-site sequence with linking experiments showed that the human the U2 snRNP, and ®nally, association of the U4/ 220 kDa protein and its yeast homologue Prp8p U6.U5tri-snRNP.2,3However,severalrecentdata cover large parts of the 50-stem-loop structure of suggestthattheassemblyprocessismorecomplex U5snRNA.43,44Itisalsowellestablishedthatthe thanpreviouslyproposed.6,7Bothspliceosome common set of Sm proteins binds the Sm site of U4 assembly and catalysis involve a dynamic network andU5snRNAs.45,46Atleast,threesetsoftri- of snRNA/snRNA and snRNA/premRNA inter- snRNP proteins (Sm, Lsm and some of the proteins actions.3,8,9Inparticular,integrationoftheU4/U6. cited above) were found to form multi-protein U5 tri-snRNP into the pre-spliceosomal complex complexes.35,47±50 initiates a complete remodeling of the RNA/RNA To obtain more information on UsnRNA/protein interactions in this complex: the U4/U6 interaction and RNA/RNA interactions in the yeast and is disrupted, allowing U6 snRNA to interact with human U4/U6.U5 tri-snRNPs, we performed foot- the50regionofU2snRNA,10±13theinitialinter- printing experiments on UsnRNAs in these par- action of U1 snRNA with the 50 splice site is also ticles. Here, we describe a comparative study of disrupted, and a short base-pair interaction is the U4, U5 and U6 snRNA structures and their formed between the highly conserved ACAGAG accessibility in tri-snRNP particles that were puri- sequence of U6 snRNA and the 50 extremity of the ®ed at low salt concentration from HeLa nuclear intron.14±21EstablishmentofthisU6/50splicesite extract and Saccharomyces cerevisiae cellular extra- interaction is thought to be a prerequisite for U4/ ct.28,30Thisstudyisbasedontheuseofseveral U6dissociation22andU5snRNAislikelyinvolved chemical reagents and RNases under conditions in this process by formation of a U5/50 splice site that were selected to preserve the tri-snRNP struc- interactionearlyinassembly.3,9,23Priortothe®rst ture. For a better identi®cation of the UsnRNA seg- step of the reaction, U5 snRNA interacts with the ments protected by proteins, analyses were 30 extremity of the upstream exon. After this ®rst performed under the same conditions on naked step, it interacts with the two exon extremities, that yeast and human UsnRNAs, the human U4/U6 havetobeligated.6,16,18,24±26Thiscascadeofremo- snRNA duplex and the human U4/U6 10 S snRNP deling of the RNA/RNA interactions, that is puri®ed at high salt concentration by MonoQ initiated upon tri-snRNP integration, is highly con- chromatography.51Protectionwasfoundtobe served from yeast to vertebrates. This strongly stronger in the human tri-snRNP than in the yeast suggests that UsnRNA structures and RNA/pro- tri-snRNP. The possible identity of components tein interactions in the tri-snRNP have been that afforded the protection is discussed, taking selected for a correct achievement of all these into account present knowledge on UsnRNAs and RNA-strand exchanges. In addition, tri-snRNP pro- tri-snRNPproteins.30,31,34,78 teins also play a crucial role in these exchanges, as demonstratedforsomeofthem.27Hence,aprecise understanding of the tri-snRNP architecture is Results required for a complete elucidation of the mechan- Strategy used in this study isms responsible for the structural transitions lead- ing to active splicing complexes. The protein To identify the segments of UsnRNAs that are content of the tri-snRNP particle is now well estab- protected by proteins in the tri-snRNPs, we had to lishedforthehumanandyeastsystems.28±31 use conditions for RNA probing that were as About 30 distinct proteins were identi®ed in the gentle as possible to maintain the structural integ- puri®ed human tri-snRNP, many of which are rity of human and yeast tri-snRNP particles. We essentialforsplicing.32Homologuesofmany had previously developed conditions to probe the human tri-snRNP proteins were found in the puri- U3snoRNAstructureintheyeastU3snoRNP.52 ®edyeasttri-snRNP.30,31Someofthesehighlycon- Starting from these established conditions, we served proteins, with speci®c catalytic activities modi®ed the concentrations of reagents and (ATPase, GTPase, proline isomerase) have been the enzymes, as well as times of incubation, in order to subjectofdeepanalyses.3,33,34However,littleis limit tri-snRNP dissociation. The integrity of the known on the protein/protein and RNA/protein human and yeast 25 S tri-snRNPs was tested by interactions that bridge the U4/U6 and U5 snRNP gel electrophoresis and glycerol-gradient fraction- componentswithinthetri-snRNP,35andknowl- ation. Examples of fractionation by electrophoresis edge on the binding sites of proteins on U4/U6 under non-denaturing conditions of the human and U5 snRNAs remains limited also. Essentially, 25 S tri-snRNPs modi®ed by CMCT or DMS are the yeast Prp6 and its human homologue 102 kDa showninFigure1(a). may bridge the U4/U6 and the U5 components of To identify the segments of UsnRNAs that are the tri-snRNP; the yeast Prp3 and Prp4 proteins protected by the proteins in the tri-snRNPs, we (human homologues 60 and 90 kDa proteins) had to compare the accessibility in the particles to associate with the yeast 50-terminal stem-loop that in naked UsnRNAs and the U4/U6 snRNA structureofU4snRNA,3638thehuman15.5kDa duplex. To this end, we used a non-denaturing proteinanditsyeasthomologueSnu13pbindthe phenol extraction procedure that allowed us to 50-terminalloopofU4snRNA,39,40andtheLsm keep a small part of the human U4/U6 RNA proteinsbindthe30endofU6snRNA.41,42Cross- duplexinanintactform(Figure1(b)).Afterphenol RNA Probing in Yeast and Human U4/U6.U5 Tri-snRNPs 633 these accessibilities on the puri®ed 25 S tri-snRNP and compared them to those in free U4, U5 and U6 snRNAs. Each probing experiment was repeated between two and ®ve times, depending on the dif- ®culty to get a very clear and reproducible picture of the accessibilities. A high protection of U5 snRNA in the human 25 S tri-snRNP Representative examples of primer extension analyses of U5 snRNA modi®ed by DMS or CMCT, in intact tri-snRNP or after phenol extrac- tion,areshowninFigure2(a).Resultsobtained from different experiments are summarized in Figure2(b)and(c).Dataforbothchemicaland Figure 1.
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