NORMAL GENOMIC

• Nonomura K et al.
• A novel RNA-recognition-motif protein is required for premeioticG1/S-phase transition in rice (Oryza sativa L.).
• PLoS Genet. 2011; 7: 1001265-1001265
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• The molecular mechanism for meiotic entry remains largely elusive inflowering plants. Only Arabidopsis SWI1/DYAD and maize AM1, both of whichare the coiled-coil protein, are known to be required for the initiationof plant meiosis. The mechanism underlying the synchrony of male meiosis,characteristic to flowering plants, has also been unclear in the plantkingdom. In other eukaryotes, RNA-recognition-motif (RRM) proteins areknown to play essential roles in germ-cell development and meiosisprogression. Rice MEL2 protein discovered in this study shows partialsimilarity with human proline-rich RRM protein, deleted inAzoospermia-Associated Protein1 (DAZAP1), though MEL2 also possessesankyrin repeats and a RING finger motif. Expression analyses of severalcell-cycle markers revealed that, in mel2 mutant anthers, most germ cellsfailed to enter premeiotic S-phase and meiosis, and a part escaped fromthe defect and underwent meiosis with a significant delay or continuedmitotic cycles. Immunofluorescent detection revealed that T7peptide-tagged MEL2 localized at cytoplasmic perinuclear region of germcells during premeiotic interphase in transgenic rice plants. This studyis the first report of the plant RRM protein, which is required forregulating the premeiotic G1/S-phase transition of male and female germcells and also establishing synchrony of male meiosis. This study willcontribute to elucidation of similarities and diversities in reproductionsystem between plants and other species.

• Thickman KR, Sickmier EA, Kielkopf CL
• Alternative conformations at the RNA-binding surface of the N-terminalU2AF(65) RNA recognition motif.
• J Mol Biol. 2007; 366: 703-10
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• The essential pre-mRNA splicing factor, U2 auxiliary factor 65KD(U2AF(65)) recognizes the polypyrimidine tract (Py-tract) consensussequence of the pre-mRNA using two RNA recognition motifs (RRMs), the mostprevalent class of eukaryotic RNA-binding domain. The Py-tracts of highereukaryotic pre-mRNAs are often interrupted with purines, yet U2AF(65) mustidentify these degenerate Py-tracts for accurate pre-mRNA splicing.Previously, the structure of a U2AF(65) variant in complex with poly(U)RNA suggested that rearrangement of flexible side-chains or bound watermolecules may contribute to degenerate Py-tract recognition by U2AF(65).Here, the X-ray structure of the N-terminal RRM domain of U2AF(65) (RRM1)is described at 1.47 A resolution in the absence of RNA. Notably,RNA-binding by U2AF(65) selectively stabilizes pre-existing alternativeconformations of three side-chains located at the RNA interface (Arg150,Lys225, and Arg227). Additionally, a flexible loop connecting thebeta2/beta3 strands undergoes a conformational change to interact with theRNA. These pre-existing alternative conformations may contribute to theability of U2AF(65) to recognize a variety of Py-tract sequences. Thisrare, high-resolution view of an important member of the RRM class ofRNA-binding domains highlights the role of alternative side-chainconformations in RNA recognition.

• Tintaru AM et al.
• Structural and functional analysis of RNA and TAP binding to SF2/ASF.
• EMBO Rep. 2007; 8: 756-62
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• The serine/arginine-rich (SR) protein splicing factor 2/alternativesplicing factor (SF2/ASF) has a role in splicing, stability, export andtranslation of messenger RNA. Here, we present the structure of the RNArecognition motif (RRM) 2 from SF2/ASF, which has an RRM fold with aconsiderably extended loop 5 region, containing a two-stranded beta-sheet.The loop 5 extension places the previously identified SR protein kinase 1docking sequence largely within the RRM fold. We show that RRM2 binds toRNA in a new way, by using a tryptophan within a conserved SWQLKD motifthat resides on helix alpha1, together with amino acids from strand beta2and a histidine on loop 5. The linker connecting RRM1 and RRM2 containsarginine residues, which provide a binding site for the mRNA export factorTAP, and when TAP binds to this region it displaces RNA bound to RRM2.

• 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
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• 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.

• Law MJ, Chambers EJ, Katsamba PS, Haworth IS, Laird-Offringa IA
• Kinetic analysis of the role of the tyrosine 13, phenylalanine 56 andglutamine 54 network in the U1A/U1 hairpin II interaction.
• Nucleic Acids Res. 2005; 33: 2917-28
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• The A protein of the U1 small nuclear ribonucleoprotein particle,interacting with its stem-loop RNA target (U1hpII), is frequently used asa paradigm for RNA binding by recognition motif domains (RRMs). U1A/U1hpIIcomplex formation has been proposed to consist of at least two steps:electrostatically mediated alignment of both molecules followed by lockinginto place, based on the establishment of close-range interactions. Thesequence of events between alignment and locking remains obscure. Here weexamine the roles of three critical residues, Tyr13, Phe56 and Gln54, incomplex formation and stability using Biacore. Our mutational and kineticdata suggest that Tyr13 plays a more important role than Phe56 in complexformation. Mutational analysis of Gln54, combined with molecular dynamicsstudies, points to Arg52 as another key residue in association. Based onour data and previous structural and modeling studies, we propose thatelectrostatic alignment of the molecules is followed by hydrogen bondformation between the RNA and Arg52, and the sequential establishment ofinteractions with loop bases (including Tyr13). A quadruple stack,sandwiching two bases between Phe56 and Asp92, would occur last andcoincide with the rearrangement of a C-terminal helix that partiallyoccludes the RRM surface in the free protein.

• James SA, Turner W, Schwer B
• How Slu7 and Prp18 cooperate in the second step of yeast pre-mRNAsplicing.
• RNA. 2002; 8: 1068-77
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• Slu7 and Prp18 act in concert during the second step of yeast pre-mRNAsplicing. Here we show that the 382-amino-acid Slu7 protein contains twofunctionally important domains: a zinc knuckle (122CRNCGEAGHKEKDC135) anda Prp18-interaction domain (215EIELMKLELY224). Alanine cluster mutationsof 215EIE217 and 221LELY224 abrogated Slu7 binding to Prp18 in atwo-hybrid assay and in vitro, and elicited temperature-sensitive growthphenotypes in vivo. Yet, the mutations had no impact on Slu7 function inpre-mRNA splicing in vitro. Single alanine mutations of zinc knuckleresidues Cys122, His130, and Cys135 had no effect on cell growth, butcaused Slu7 function during pre-mRNA splicing in vitro to become dependenton Prp18. Specifically, zinc knuckle mutants required Prp18 in order tobind to the spliceosome. Compound mutations in both Slu7 domains (e.g.,C122A-EIE, H130A-EIE, and C135A-EIE) were lethal in vivo and abolishedsplicing in vitro, suggesting that the physical interaction between Slu7and Prp18 is important for cooperation in splicing.Depletion/reconstitution studies coupled with immunoprecipitations suggestthat second step factors are recruited to the spliceosome in the followingorder: Slu7 --> Prp18 --> Prp22. All three proteins are released from thespliceosome after step 2 concomitant with release of mature mRNA.

• Soderberg M, Raffalli-Mathieu F, Lang MA
• Inflammation modulates the interaction of heterogeneous nuclearribonucleoprotein (hnRNP) I/polypyrimidine tract binding protein and hnRNPL with the 3'untranslated region of the murine inducible nitric-oxidesynthase mRNA.
• Mol Pharmacol. 2002; 62: 423-31
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• Interaction of two members of the heterogeneous nuclear ribonucleoprotein(hnRNP) family with the 3'untranslated region (UTR) of the murineinducible nitric-oxide synthase (iNOS) mRNA is demonstrated in this study.An iNOS RNA-protein complex is formed using protein extracts fromuntreated and septic shock treated mouse liver. UV cross-linking revealsthat the complex consists of at least two proteins, with apparentmolecular masses of 60 and 70 kDa, respectively. The 60-kDa proteinbinding site lies within a 112-nt pyrimidine-rich sequence, approximately160 nt from the coding sequence, and the RNA-protein complex can beprecipitated by a monoclonal antibody directed against hnRNP I [also namedpolypyrimidine tract binding protein (PTB)]. The 70-kDa protein binds a43-nt sequence near the 3'end of the 3'UTR and is immunoprecipitated by amonoclonal antibody against hnRNP L. A computer-simulated conformation ofthe 3'UTR suggests that both binding sites reside in regions easilyaccessible for a protein. Supershifts of the native RNA-protein complexcould only be achieved with anti-hnRNP L, suggesting that within thismultiprotein RNA complex, only hnRNP L is exposed to the antibodies,whereas the hnRNP I/PTB is mainly responsible for its interaction with themRNA. Up-regulation of iNOS by septic shock reduces the RNA-proteincomplex formation, thus showing that hnRNP I/PTB and hnRNP L binding tothe iNOS mRNA is modulated by inflammation. This suggests a novel functionfor the two previously described proteins as regulators of the iNOS gene.

• Adams DJ, van der Weyden L, Mayeda A, Stamm S, Morris BJ, Rasko JE
• ZNF265--a novel spliceosomal protein able to induce alternative splicing.
• J Cell Biol. 2001; 154: 25-32
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• The formation of the active spliceosome, its recruitment to active areasof transcription, and its role in pre-mRNA splicing depends on theassociation of a number of multifunctional serine/arginine-rich (SR)proteins. ZNF265 is an arginine/serine-rich (RS) domain containing zincfinger protein with conserved pre-mRNA splicing protein motifs. Here weshow that ZNF265 immunoprecipitates from splicing extracts in associationwith mRNA, and that it is able to alter splicing patterns of Tra2-beta1transcripts in a dose-dependent manner in HEK 293 cells. Yeast two-hybridanalysis and immunoprecipitation indicated interaction of ZNF265 with theessential splicing factor proteins U1-70K and U2AF(35). Confocalmicroscopy demonstrated colocalization of ZNF265 with the motor neurongene product SMN, the snRNP protein U1-70K, the SR protein SC35, and withthe transcriptosomal components p300 and YY1. Transfection of HT-1080cells with ZNF265-EGFP fusion constructs showed that nuclear localizationof ZNF265 required the RS domain. Alignment with other RSdomain-containing proteins revealed a high degree of SR dipeptideconservation. These data show that ZNF265 functions as a novel componentof the mRNA processing machinery.

• 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
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• 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.

• Welin Henriksson E, Wahren-Herlenius M, Lundberg I, Mellquist E, Pettersson I
• Key residues revealed in a major conformational epitope of the U1-70Kprotein.
• Proc Natl Acad Sci U S A. 1999; 96: 14487-92
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• Epitopes depending on three-dimensional folding of proteins have duringrecent years been acknowledged to be main targets for many autoantibodies.However, a detailed resolution of conformation-dependent epitopes has todate not been achieved in spite of its importance for understanding thecomplex interaction between an autoantigen and the immune system. Inanalysis of immunodominant epitopes of the U1-70K protein, the majorautoantigen recognized by human ribonucleoprotein (RNP)-positive sera, wehave used diversely mutated recombinant Drosophila melanogaster 70Kproteins as antigens in assays for human anti-RNP antibodies. Thus, thecontribution of individual amino acids to antigenicity could be assayedwith the overall structure of the major antigenic domain preserved, andanalysis of how antigenicity can be reconstituted rather than obliteratedwas enabled. Our results reveal that amino acid residue 125 is situated ata crucial position for recognition by human anti-RNP autoantibodies andthat flanking residues at positions 119-126 also appear to be of utmostimportance for recognition. These results are discussed in relation tostructural models of RNA-binding domains, and tertiary structure modelingindicates that the residues 119-126 are situated at easily accessiblepositions in the end of an alpha-helix in the RNA binding region. Thisstudy identifies a major conformation-dependent epitope of the U1-70Kprotein and demonstrates the significance of individual amino acids inconformational epitopes. Using this model, we believe it will be possibleto analyze other immunodominant regions in which protein conformation hasa strong impact.

• Zorio DA, Blumenthal T
• U2AF35 is encoded by an essential gene clustered in an operon withRRM/cyclophilin in Caenorhabditis elegans.
• RNA. 1999; 5: 487-94
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• In most species the 3' splice site is recognized initially by aninteraction between the two-subunit splicing factor U2AF with thepolypyrimidine (poly(Y)) tract that results in recruitment of the U2 snRNPto the branch-point consensus just upstream. In contrast, inCaenorhabditis elegans, both the poly(Y) tract and the branch-pointconsensus sequences are missing, apparently replaced by the highlyconserved U4CAG/R 3' splice site consensus. Nevertheless C. elegans U2AF65is very similar to its mammalian and fly counterparts and may recognizethe 3' splice site consensus. Here we report the cloning of the C. elegansU2AF35 gene, uaf-2. We show that it lacks an identifiable RS domain,which, in flies, has been shown to play a role in RNA binding, but itcontains an extended glycine-rich stretch at its C-terminus. uaf-2 is inan operon with cyp-13, a gene that encodes a cyclophilin with an RRMdomain at its N-terminus. We demonstrate by RNA interference that bothU2AF genes, uaf-1 (which encodes U2AF65) and uaf-2, are required forviability, whereas cyp-13 is apparently not.

• Shen EC, Henry MF, Weiss VH, Valentini SR, Silver PA, Lee MS
• Arginine methylation facilitates the nuclear export of hnRNP proteins.
• Genes Dev. 1998; 12: 679-91
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• Eukaryotic mRNA processing and export is mediated by various heterogeneousnuclear ribonucleoproteins (hnRNPs). Many of these hnRNPs are methylatedon arginine residues. In the yeast, Saccharomyces cerevisiae, thepredominant enzyme responsible for arginine methylation is Hmt1p. Hmt1pmethylates both Npl3p and Hrp1p, which are shuttling hnRNPs involved inmRNA processing and export. Here, we employ an in vivo nuclear exportassay to show that arginine methylation is important for the nuclearexport of these hnRNPs. Both Npl3p and Hrp1p fail to exit the nucleus incells lacking Hmt1p, and overexpression of Hmt1p enhances Npl3p export.The export of a novel hnRNP-like protein, Hrb1p, which does not bindpoly(A)+ RNA, however, is not affected by the lack of methylation.Furthermore, we find a genetic relationship between Hmt1p and cap-bindingprotein 80 (CBP80). Together, these findings establish that one biologicalrole for arginine methylation is in facilitating the export of certainhnRNPs out of the nucleus.

• Abe R, Yamamoto K, Sakamoto H
• Target specificity of neuronal RNA-binding protein, Mel-N1: direct bindingto the 3' untranslated region of its own mRNA.
• Nucleic Acids Res. 1996; 24: 2011-6
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• We have identified cDNAs encoding Mel-N1, the mouse homologue of a humannervous system-specific RNA-binding protein, Hel-N1. Two major mRNAtranscripts of Mel-N1 were detected predominantly in the adult mouse brainby Northern blot analysis. To gain insight into the RNA bindingspecificity of Mel-N1, we performed iterative in vitro RNA selection. Theresulting in vitro selected RNAs were found to contain AU-rich sequencesas well as a GAAA motif in the majority of clones. By means of in vitrobinding assays we demonstrate that this GAAA sequence appears tosignificantly affect the Mel-N1 RNA-binding efficiency. Our studiesfurther reveal that Mel-N1 can bind to its own 3' untranslated region(3'UTR) as well as to the c-fos 3'UTR, and is localized predominantly inthe cytoplasmic region in cells, suggesting that posttranscriptionalautoregulation of Mel-N1 gene expression occurs in vivo.

• Lin JJ, Zakian VA
• Isolation and characterization of two Saccharomyces cerevisiae genes thatencode proteins that bind to (TG1-3)n single strand telomeric DNA invitro.
• Nucleic Acids Res. 1994; 22: 4906-13
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• By screening lambda gt11 libraries with a radiolabeled (TG1-3)noligonucleotide, two Saccharomyces cerevisiae genes were identified thatencode polypeptides that recognize the single-stranded telomeric repeatsequence (TG1-3)n. The first gene, NSR1, a previously identified gene,encodes a protein involved in ribosomal RNA maturation and possibly intransport of proteins into the nucleus. The second gene, GBP2 (G-strandBinding Protein), is an anonymous open reading frame from chromosome III.These two genes contain RNA recognition motifs (RRMs) that are found inproteins that interact with RNA. Both Nsr1p and Gbp2p bind specifically toyeast single strand (TG1-3)n DNA in vitro. To test whether these twoproteins associate with telomeres in vivo, strains were constructed inwhich one or both of these genes were either disrupted or overexpressed.None of these alterations affected telomere length or telomere positioneffect. The potential role of these two (TG1-3)n binding proteins isdiscussed.

• Zahler AM, Neugebauer KM, Lane WS, Roth MB
• Distinct functions of SR proteins in alternative pre-mRNA splicing.
• Science. 1993; 260: 219-22
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• Alternative splicing of precursor messenger RNAs (pre-mRNAs) is a commonmechanism of regulating gene expression. SR proteins are a family ofpre-mRNA splicing factors that are structurally related and evolutionarilyconserved. Any member of the SR family can complement a splicing-deficientextract that lacks the entire family of SR proteins. Here it isdemonstrated that particular SR proteins have distinct functions inalternative pre-mRNA splicing in vitro. In addition, SR proteins aredifferentially expressed in a variety of tissues. These results suggest afundamental role for SR proteins in the regulation of alternativesplicing.

• Vellard M, Sureau A, Soret J, Martinerie C, Perbal B
• A potential splicing factor is encoded by the opposite strand of thetrans-spliced c-myb exon.
• Proc Natl Acad Sci U S A. 1992; 89: 2511-5
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• We previously established that the expression of a thymic c-myb mRNAspecies requires the intermolecular recombination of coding sequencesexpressed from transcriptional units localized on different chromosomes,in both chicken and human. We now report that a putative splicing factor(PR264), extremely well conserved in chicken and human, is encoded by theopposite strand of the c-myb trans-spliced exon. The PR264 polypeptide,which contains a typical ribonucleoprotein 80 and an arginine/serine-richdomain, is highly homologous to the Drosophila splicing regulators tra,tra-2, and su(wa) and to the human alternative splicing factor ASF/SF2.Furthermore, we show that PR264-specific mRNAs are expressed in normalhematopoietic cells of chicken and human origin and that the relativeproportion of the PR264 transcripts is developmentally regulated inchicken.

• Krainer AR, Mayeda A, Kozak D, Binns G
• Functional expression of cloned human splicing factor SF2: homology toRNA-binding proteins, U1 70K, and Drosophila splicing regulators.
• Cell. 1991; 66: 383-94
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• SF2 is a protein factor essential for constitutive pre-mRNA splicing inHeLa cell extracts and also activates proximal alternative 5' splice sitesin a concentration-dependent manner. This latter property suggests a rolefor SF2 in preventing exon skipping, ensuring the accuracy of splicing,and regulating alternative splicing. Human SF2 cDNAs have been isolatedand overexpressed in bacteria. Recombinant SF2 is active in splicing andstimulates proximal 5' splice sites. SF2 has a C-terminal region rich inarginine-serine dipeptides, similar to the RS domains of the U1 snRNP 70Kpolypeptide and the Drosophila alternative splicing regulatorstransformer, transformer-2, and suppressor-of-white-apricot. Liketransformer-2 and 70K, SF2 contains an RNP-type RNA recognition motif.

• Ayane M, Preuss U, Kohler G, Nielsen PJ
• A differentially expressed murine RNA encoding a protein with similaritiesto two types of nucleic acid binding motifs.
• Nucleic Acids Res. 1991; 19: 1273-8
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• Using differential screening, a murine cDNA, termed X16, was isolatedcorresponding to an mRNA which is more strongly expressed in pre-B celllines relative to mature B-cell lines. The complete coding sequence of themRNA predicts a 19kD protein with two domains connected by a proline-richspacer. The N-terminal domain of about 90 amino acids encodes an RNAbinding motif including the ribonucleoprotein consensus octapeptide foundin one class of RNA-binding proteins and highly conserved from yeast toman. Within the very basic C-terminal domain of about 60 amino acids,several copies of two different peptides are found which are also presentin several proteins which bind DNA or RNA. The expression of X16 is notlimited to the lymphoid lineage. In adult mice, although the strongestexpression was seen in thymus, mRNA was also found in testis, brain,spleen, and very low in heart. X16 mRNA was not detected in liver andkidney. In tissue culture, the expression of X16 mRNA can be induced byserum. The conserved protein motifs and expression pattern suggest thatX16 could be involved in RNA processing correlating with cellularproliferation.

• Ge H, Zuo P, Manley JL
• Primary structure of the human splicing factor ASF reveals similaritieswith Drosophila regulators.
• Cell. 1991; 66: 373-82
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• We described previously the purification of a human protein, calledalternative splicing factor (ASF), that can switch utilization ofalternative 5' splice sites in an SV40 early pre-mRNA. We now report theisolation of a cDNA, designated ASF-1, that encodes this protein. ASF-1consists of 248 amino acid residues, including an 80 residue RNA-bindingdomain at its N-terminus and a 50 residue C-terminal region that is 80%serine plus arginine. ASF-1 produced in E. coli can activate splicing invitro and switch 5' splice-site utilization, establishing that therecombinant protein is sufficient to supply these activities. Analysis ofadditional cDNAs revealed that ASF pre-mRNA can itself be alternativelyspliced, surprisingly, by utilization of a shared 5' splice site and twoclosely spaced 3' splice sites. Use of the upstream site results in asecond mRNA (ASF-2) in which translation of the downstream exon occursextensively in an alternative reading frame distinct from ASF-1.

• Bentley RC, Keene JD
• Recognition of U1 and U2 small nuclear RNAs can be altered by a5-amino-acid segment in the U2 small nuclear ribonucleoprotein particle(snRNP) B" protein and through interactions with U2 snRNP-A' protein.
• Mol Cell Biol. 1991; 11: 1829-39
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• We have investigated the sequence elements influencing RNA recognition intwo closely related small nuclear ribonucleoprotein particle (snRNP)proteins, U1 snRNP-A and U2 snRNP-B". A 5-amino-acid segment in theRNA-binding domain of the U2 snRNP-B" protein was found to confer U2 RNArecognition when substituted into the corresponding position in the U1snRNP-A protein. In addition, B", but not A, was found to require the U2snRNP-A' protein as an accessory factor for high-affinity binding to U2RNA. The pentamer segment in B" that conferred U2 RNA recognition was notsufficient to allow the A' enhancement of U2 RNA binding by B", thusimplicating other sequences in this protein-protein interaction. Sequenceelements involved in these interactions have been localized to variableloops of the RNA-binding domain as determined by nuclear magneticresonance spectroscopy (D. Hoffman, C.C. Query, B. Golden, S.W. White, andJ.D. Keene, Proc. Natl. Acad. Sci. USA, in press). These findings suggesta role for accessory proteins in the formation of RNP complexes andpinpoint amino acid sequences that affect the specificity of RNArecognition in two members of a large family of proteins involved in RNAprocessing.

• Nietfeld W, Mentzel H, Pieler T
• The Xenopus laevis poly(A) binding protein is composed of multiplefunctionally independent RNA binding domains.
• EMBO J. 1990; 9: 3699-705
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• A family of eukaryotic RNA binding proteins is defined by the conservedRNP motif. The poly(A) binding protein has four such motifs. We report onthe isolation and structural characterization of several variant cDNAclones, as well as of a gene encoding this protein in Xenopus laevisembryos. Wild-type protein as well as truncated versions carrying isolatedsingle motifs or artificial combinations of two and more such elementswere characterized for their ability to bind specifically to RNAhomopolymers. Three of the isolated repeats were functional in specificRNA binding, whereas the N-terminal RNP motif was non-functional.Combinatorial effects in RNA binding were measured with constructscarrying multiple repeats, which were not predictable from the activity ofisolated domains.

• Kumar A et al.
• Mammalian heterogeneous nuclear ribonucleoprotein A1. Nucleic acid bindingproperties of the COOH-terminal domain.
• J Biol Chem. 1990; 265: 17094-100
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• A1 is a core protein of the eukaryotic heterogeneous nuclearribonucleoprotein complex and is under study here as a prototypesingle-stranded nucleic acid-binding protein. A1 is a two-domain protein,NH2-terminal and COOH-terminal, with highly conserved primary structureamong vertebrate homologues sequenced to date. It is well documented thatthe NH2-terminal domain has single-stranded DNA and RNA binding activity.We prepared a proteolytic fragment of rat A1 representing theCOOH-terminal one-third of the intact protein, the region previouslytermed COOH-terminal domain. This purified fragment of 133 amino acidsbinds to DNA and also binds tightly to the fluorescent reporterpoly(ethenoadenylate), which is used to access binding parameters. Insolution with 0.41 M NaCl, the equilibrium constant is similar to thatobserved with A1 itself, and binding is cooperative. The purifiedCOOH-terminal fragment can be photochemically cross-linked to boundnucleic acid, confirming that COOH-terminal fragment residues are in closecontact with the polynucleotide lattice. These binding results withisolated COOH-terminal fragment indicate that the COOH-terminal domain inintact A1 can contribute directly to binding properties. Contact betweenboth COOH-terminal domain and NH2-terminal domain residues in an intactA1:poly(8-azidoadenylate) complex was confirmed by photochemicalcross-linking.

• Surowy CS, van Santen VL, Scheib-Wixted SM, Spritz RA
• Direct, sequence-specific binding of the human U1-70K ribonucleoproteinantigen protein to loop I of U1 small nuclear RNA.
• Mol Cell Biol. 1989; 9: 4179-86
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• We have studied the interaction of two of the U1 small nuclearribonucleoprotein (snRNP)-specific proteins, U1-70K and U1-A, with U1small nuclear RNA (snRNA). The U1-70K protein is a U1-specific RNA-bindingprotein. Deletion and mutation analyses of a beta-galactosidase/U1-70Kpartial fusion protein indicated that the central portion of the protein,including the RNP sequence domain, is both necessary and sufficient forspecific U1 snRNA binding in vitro. The highly conserved eight-amino-acidRNP consensus sequence was found to be essential for binding. Deletion andmutation analyses of U1 snRNA showed that both the U1-70K fusion proteinand the native HeLa U1-70K protein bound directly to loop I of U1 snRNA.Binding was sequence specific, requiring 8 of the 10 bases in the loop.The U1-A snRNP protein also interacted specifically with U1 snRNA,principally with stem-loop II.