NORMAL GENOMIC

• Golovanov AP, Hautbergue GM, Tintaru AM, Lian LY, Wilson SA
• The solution structure of REF2-I reveals interdomain interactions andregions involved in binding mRNA export factors and RNA.
• RNA. 2006; 12: 1933-48
• Display abstract

• The RNA binding and export factor (REF) family of mRNA export adaptors arefound in several nuclear protein complexes including the spliceosome,TREX, and exon junction complexes. They bind RNA, interact with thehelicase UAP56/DDX39, and are thought to bridge the interaction betweenthe export factor TAP/NXF1 and mRNA. REF2-I consists of three domains,with the RNA recognition motif (RRM) domain positioned in the middle. Herewe dissect the interdomain interactions of REF2-I and present the solutionstructure of a functionally competent double domain (NM; residues 1-155).The N-terminal domain comprises a transient helix (N-helix) linked to theRRM by a flexible arm that includes an Arg-rich region. The N-helix, whichis required for REF2-I function in vivo, overlaps the highly conservedREF-N motif and, together with the adjacent Arg-rich region, interactstransiently with the RRM. RNA interacts with REF2-I through arginine-richregions in its N- and C-terminal domains, but we show that it alsointeracts weakly with the RRM. The mode of interaction is unusual for anRRM since it involves loops L1 and L5. NMR signal mapping and biochemicalanalysis with NM indicate that DDX39 and TAP interact with both the N andRRM domains of REF2-I and show that binding of these proteins and RNA willfavor an open conformation for the two domains. The proximity of the RNA,TAP, and DDX39 binding sites on REF2-I suggests their binding may bemutually exclusive, which would lead to successive ligand binding eventsin the course of mRNA export.

• Leulliot N et al.
• A new alpha-helical extension promotes RNA binding by the dsRBD of Rnt1pRNAse III.
• EMBO J. 2004; 23: 2468-77
• Display abstract

• Rnt1 endoribonuclease, the yeast homolog of RNAse III, plays an importantrole in the maturation of a diverse set of RNAs. The enzymatic activityrequires a conserved catalytic domain, while RNA binding requires thedouble-stranded RNA-binding domain (dsRBD) at the C-terminus of theprotein. While bacterial RNAse III enzymes cleave double-stranded RNA,Rnt1p specifically cleaves RNAs that possess short irregular stem-loopscontaining 12-14 base pairs interrupted by internal loops and bulges andcapped by conserved AGNN tetraloops. Consistent with this substratespecificity, the isolated Rnt1p dsRBD and the 30-40 amino acids thatfollow bind to AGNN-containing stem-loops preferentially in vitro. Inorder to understand how Rnt1p recognizes its cognate processing sites, wehave defined its minimal RNA-binding domain and determined its structureby solution NMR spectroscopy and X-ray crystallography. We observe a newcarboxy-terminal helix following a canonical dsRBD structure. Removal ofthis helix reduces binding to Rnt1p substrates. The results suggest thatthis helix allows the Rnt1p dsRBD to bind to short RNA stem-loops bymodulating the conformation of helix alpha1, a key RNA-recognition elementof the dsRBD.

• Ramos A, Hollingworth D, Pastore A
• The role of a clinically important mutation in the fold and RNA-bindingproperties of KH motifs.
• RNA. 2003; 9: 293-8
• Display abstract

• We have investigated the role in the fold and RNA-binding properties ofthe KH modules of a hydrophobic to asparagine mutation of clinicalimportance in the fragile X syndrome. The mutation involves awell-conserved hydrophobic residue close to the N terminus of the secondhelix of the KH fold (alpha2(3) position). The effect of the mutation hasbeen long debated: Although the mutant has been shown to disrupt thethree-dimensional fold of several KH domains, the residue seems also to bedirectly involved in RNA binding, the main function of the KH module. Herewe have used the KH3 of Nova-1, whose structure is known both in isolationand in an RNA complex, to study in detail the role of the alpha2(3)position. A detailed comparison of Nova KH3 structure with its RNA/KHcomplex and with other KH structures suggests a dual role for thealpha2(3) residue, which is involved both in stabilizing the hydrophobiccore and in RNA contacts. We further show by nuclear magnetic resonance(NMR) studies in solution that L447 of Nova-1 in position alpha2(3) is inexchange in the absence of RNA, and becomes locked in a more rigidconformation only upon formation of an RNA complex. This implies thatposition alpha2(3) functions as a "gate" in the mechanism of RNArecognition of KH motifs based on the rigidification of the fold upon RNAbinding.

• Suzuki M, Jeong SY, Karbowski M, Youle RJ, Tjandra N
• The solution structure of human mitochondria fission protein Fis1 revealsa novel TPR-like helix bundle.
• J Mol Biol. 2003; 334: 445-58
• Display abstract

• Fis1 in yeast localizes to the outer mitochondrial membrane andfacilitates mitochondrial fission by forming protein complexes with Dnm1and Mdv1. Fis1 orthologs exist in higher eukaryotes, suggesting that theyare functionally conserved. In the present study, we cloned the human Fis1ortholog that was predicted in a database, and determined the proteinstructure using NMR spectroscopy. Following a flexible N-terminal tail,six alpha-helices connected with short loops construct a single coredomain. The C-terminal tail containing a transmembrane segment appears tobe disordered. In the core domain, each of two sequentially adjacenthelices forms a hairpin-like conformation, resulting in a six helixassembly forming a slightly twisted slab similar to that of a tandem arrayof tetratrico-peptide repeat (TPR) motif folds. Within this TPR-like coredomain, no significant sequence similarity to the typical TPR motif isfound. The structural analogy to the TPR-containing proteins suggests thatFis1 binds to other proteins at its concave hydrophobic surface. A simplecomposition of Fis1 comprised of a binding domain and a transmembranesegment indicates that the protein may function as a molecular adaptor onthe mitochondrial outer membrane. In HeLa cells, however, increased levelsin mitochondria-associated Fis1 did not result in mitochondrialtranslocation of Drp1, a potential binding partner of Fis1 implicated inthe regulation of mitochondrial fission, suggesting that the interactionbetween Drp1 and Fis1 is regulated.

• Allain FH, Bouvet P, Dieckmann T, Feigon J
• Molecular basis of sequence-specific recognition of pre-ribosomal RNA bynucleolin.
• EMBO J. 2000; 19: 6870-81
• Display abstract

• The structure of the 28 kDa complex of the first two RNA binding domains(RBDs) of nucleolin (RBD12) with an RNA stem-loop that includes thenucleolin recognition element UCCCGA in the loop was determined by NMRspectroscopy. The structure of nucleolin RBD12 with the nucleolinrecognition element (NRE) reveals that the two RBDs bind on opposite sidesof the RNA loop, forming a molecular clamp that brings the 5' and 3' endsof the recognition sequence close together and stabilizing the stem-loop.The specific interactions observed in the structure explain the sequencespecificity for the NRE sequence. Binding studies of mutant proteins andanalysis of conserved residues support the proposed interactions. The modeof interaction of the protein with the RNA and the location of theputative NRE sites suggest that nucleolin may function as an RNA chaperoneto prevent improper folding of the nascent pre-rRNA.

• Eldridge AG, Li Y, Sharp PA, Blencowe BJ
• The SRm160/300 splicing coactivator is required for exon-enhancerfunction.
• Proc Natl Acad Sci U S A. 1999; 96: 6125-30
• Display abstract

• Exonic splicing enhancer (ESE) sequences are important for the recognitionof splice sites in pre-mRNA. These sequences are bound by specificserine-arginine (SR) repeat proteins that promote the assembly of splicingcomplexes at adjacent splice sites. We have recently identified a splicing"coactivator," SRm160/300, which contains SRm160 (the SR nuclear matrixprotein of 160 kDa) and a 300-kDa nuclear matrix antigen. In the presentstudy, we show that SRm160/300 is required for a purine-rich ESE topromote the splicing of a pre-mRNA derived from the Drosophila doublesexgene. The association of SRm160/300 and U2 small nuclear ribonucleoproteinparticle (snRNP) with this pre-mRNA requires both U1 snRNP and factorsbound to the ESE. Independently of pre-mRNA, SRm160/300 specificallyinteracts with U2 snRNP and with a human homolog of the Drosophilaalternative splicing regulator Transformer 2, which binds to purine-richESEs. The results suggest a model for ESE function in which the SRm160/300splicing coactivator promotes critical interactions between ESE-bound"activators" and the snRNP machinery of the spliceosome.

• Greenbaum NL, Radhakrishnan I, Patel DJ, Hirsh D
• Solution structure of the donor site of a trans-splicing RNA.
• Structure. 1996; 4: 725-33
• Display abstract

• BACKGROUND: RNA splicing is both ubiquitous and essential for thematuration of precursor mRNA molecules in eukaryotes. The process oftrans-splicing involves the transfer of a short spliced leader (SL) RNAsequence to a consensus acceptor site on a separate pre-mRNA transcript.In Caenorhabditis elegans, a majority of pre-mRNA transcripts receive the22-nucleotide SL from the SL1 RNA. Very little is known about the variousroles that RNA structures play in the complex conformationalrearrangements and reactions involved in premRNA splicing. RESULTS: Wehave determined the solution structure of a domain of the first stem loopof the SL1 RNA of C. elegans, using homonuclear and heteronuclear NMRtechniques; this domain contains the splice-donor site and anine-nucleotide hairpin loop. In solution, the SL1 RNA fragment adopts astem-loop structure: nucleotides in the stem region form a classicalA-type helix while nucleotides in the hairpin loop specify a novelconformation that includes a helix, that extends for the first threeresidues; a syn guanosine nucleotide at the turn region; and anextrahelical adenine that defines a pocket with nucleotides at the base ofthe loop. CONCLUSION: The proximity of this pocket to the splice donorsite, combined with the observation that the nucleotides in this motif areconserved among all nematode SL RNAs, suggests that this pocket mayprovide a recognition site for a protein or RNA molecule in thetrans-splicing process.