Limited complementarity between U1 snRNA and a retroviral 5′ splice site permits its attenuation via RNA secondary structure

Multiple types of regulation are used by cells and viruses to control alternative splicing. In murine leukemia virus, accessibility of the 5′ splice site (ss) is regulated by an upstream region, which can fold into a complex RNA stem–loop structure. The underlying sequence of the structure itself is negligible, since most of it could be functionally replaced by a simple heterologous RNA stem–loop preserving the wild-type splicing pattern. Increasing the RNA duplex formation between U1 snRNA and the 5′ss by a compensatory mutation in position +6 led to enhanced splicing. Interestingly, this mutation affects splicing only in the context of the secondary structure, arguing for a dynamic interplay between structure and primary 5′ss sequence. The reduced 5′ss accessibility could also be counteracted by recruiting a splicing enhancer domain via a modified MS2 phage coat protein to a single binding site at the tip of the simple RNA stem–loop. The mechanism of 5′ss attenuation was revealed using hyperstable U1 snRNA mutants, showing that restricted U1 snRNP access is the cause of retroviral alternative splicing.

[1]  A. Kornblihtt,et al.  Multiple links between transcription and splicing. , 2004, RNA.

[2]  D. Niederacher,et al.  Diagnostics of pathogenic splicing mutations: does bioinformatics cover all bases? , 2008, Frontiers in bioscience : a journal and virtual library.

[3]  C. Burge,et al.  Weak definition of IKBKAP exon 20 leads to aberrant splicing in familial dysautonomia , 2007, Human mutation.

[4]  B. Amendt,et al.  Presence of exon splicing silencers within human immunodeficiency virus type 1 tat exon 2 and tat-rev exon 3: evidence for inhibition mediated by cellular factors , 1995, Molecular and cellular biology.

[5]  M. Garcia-Blanco,et al.  Protein–protein interactions and 5'-splice-site recognition in mammalian mRNA precursors , 1994, Nature.

[6]  M. Rosbash,et al.  Short artificial hairpins sequester splicing signals and inhibit yeast pre-mRNA splicing , 1993, Molecular and cellular biology.

[7]  A. Grover,et al.  5′ Splice Site Mutations in tau Associated with the Inherited Dementia FTDP-17 Affect a Stem-Loop Structure That Regulates Alternative Splicing of Exon 10* , 1999, The Journal of Biological Chemistry.

[8]  K. J. Hertel,et al.  Combinatorial Control of Exon Recognition* , 2008, Journal of Biological Chemistry.

[9]  P. Sharp,et al.  Complementation by SR proteins of pre-mRNA splicing reactions depleted of U1 snRNP. , 1994, Science.

[10]  C. Ehresmann,et al.  Conformational analysis of the 5' leader and the gag initiation site of Mo-MuLV RNA and allosteric transitions induced by dimerization. , 1993, Nucleic acids research.

[11]  Stephen M. Mount,et al.  The U1 small nuclear RNA-protein complex selectively binds a 5′ splice site in vitro , 1983, Cell.

[12]  N. Lemp,et al.  Adaptive evolution of a tagged chimeric gammaretrovirus: identification of novel cis-acting elements that modulate splicing. , 2007, Journal of molecular biology.

[13]  B. Peterlin,et al.  Trans-activation by HIV-1 Tat via a heterologous RNA binding protein , 1990, Cell.

[14]  M. Caputi,et al.  A Bidirectional SF2/ASF- and SRp40-Dependent Splicing Enhancer Regulates Human Immunodeficiency Virus Type 1 rev, env, vpu, and nef Gene Expression , 2004, Journal of Virology.

[15]  J. Hauber,et al.  The strength of the HIV-1 3' splice sites affects Rev function , 2006, Retrovirology.

[16]  E. Androphy,et al.  Modulating role of RNA structure in alternative splicing of a critical exon in the spinal muscular atrophy genes , 2006, Nucleic acids research.

[17]  M. Ohman,et al.  Coordination of editing and splicing of glutamate receptor pre-mRNA. , 2003, RNA.

[18]  H. Kräusslich,et al.  Splicing of human immunodeficiency virus RNA is position-dependent suggesting sequential removal of introns from the 5′ end , 2005, Nucleic acids research.

[19]  B. Séraphin,et al.  A U1 snRNA:pre‐mRNA base pairing interaction is required early in yeast spliceosome assembly but does not uniquely define the 5′ cleavage site. , 1988, The EMBO journal.

[20]  H. Kräusslich,et al.  A New RNA Element Located in the Coding Region of a Murine Endogenous Retrovirus Can Functionally Replace the Rev/Rev-Responsive Element System in Human Immunodeficiency Virus Type 1 Gag Expression , 2001, Journal of Virology.

[21]  A. Krainer,et al.  Pre-mRNA splicing in the new millennium. , 2001, Current opinion in cell biology.

[22]  K. Abel,et al.  Design of 5′ Untranslated Sequences in Retroviral Vectors Developed for Medical Use , 1999, Journal of Virology.

[23]  J. Valcárcel,et al.  Interaction of U2AF65 RS region with pre-mRNA branch point and promotion of base pairing with U2 snRNA [corrected]. , 1996, Science.

[24]  M. Emerman,et al.  Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated beta-galactosidase gene , 1992, Journal of virology.

[25]  Eric T. Wang,et al.  Alternative Isoform Regulation in Human Tissue Transcriptomes , 2008, Nature.

[26]  Saba Valadkhan,et al.  The spliceosome: a ribozyme at heart? , 2007, Biological chemistry.

[27]  M. Rosbash,et al.  The U1 snRNP protein U1C recognizes the 5′ splice site in the absence of base pairing , 2002, Nature.

[28]  C Maayan,et al.  Tissue-specific expression of a splicing mutation in the IKBKAP gene causes familial dysautonomia. , 2001, American journal of human genetics.

[29]  A. Schambach,et al.  Overcoming promoter competition in packaging cells improves production of self-inactivating retroviral vectors , 2006, Gene Therapy.

[30]  D. Solnick.  Alternative splicing caused by RNA secondary structure , 1985, Cell.

[31]  Jørgen Kjems,et al.  A novel approach to describe a U1 snRNA binding site. , 2003, Nucleic acids research.

[32]  Michael R. Green,et al.  RS domains contact splicing signals and promote splicing by a common mechanism in yeast through humans. , 2006, Genes & development.

[33]  M. Konarska,et al.  Insights into the mechanisms of splicing: more lessons from the ribosome. , 2005, Genes & development.

[34]  A. Zahler,et al.  hnRNP A/B proteins are required for inhibition of HIV‐1 pre‐mRNA splicing , 1999, The EMBO journal.

[35]  D. Ropers,et al.  Conserved stem-loop structures in the HIV-1 RNA region containing the A3 3' splice site and its cis-regulatory element: possible involvement in RNA splicing. , 2001, Nucleic acids research.

[36]  J. Manley,et al.  Factors influencing alternative splice site utilization in vivo , 1987, Molecular and cellular biology.

[37]  M. McNally,et al.  U1 Small Nuclear Ribonucleoprotein and Splicing Inhibition by the Rous Sarcoma Virus Negative Regulator of Splicing Element , 1999, Journal of Virology.

[38]  R. Sachidanandam,et al.  KRAINER splice sites ′ Determinants of the inherent strength of human 5 , 2005 .

[39]  B. Berkhout,et al.  RNA Structure Modulates Splicing Efficiency at the Human Immunodeficiency Virus Type 1 Major Splice Donor , 2007, Journal of Virology.

[40]  T. Nilsen,et al.  Dynamic Regulation of Alternative Splicing by Silencers that Modulate 5′ Splice Site Competition , 2008, Cell.

[41]  R. Lührmann,et al.  Structure-probing of U1 snRNPs gradually depleted of the U1-specific proteins A, C and 70k. Evidence that A interacts differentially with developmentally regulated mouse U1 snRNA variants. , 1990, Nucleic acids research.

[42]  A. Weiner,et al.  A compensatory base change in U1 snRNA suppresses a 5′ splice site mutation , 1986, Cell.

[43]  C. Burge,et al.  Splicing regulation: from a parts list of regulatory elements to an integrated splicing code. , 2008, RNA.

[44]  C. Will,et al.  The Spliceosome: Design Principles of a Dynamic RNP Machine , 2009, Cell.

[45]  I. Graham,et al.  Effects of RNA secondary structure on alternative splicing of Pre-mRNA: Is folding limited to a region behind the transcribing RNA polymerase? , 1988, Cell.

[46]  D. Black.  Mechanisms of alternative pre-messenger RNA splicing. , 2003, Annual review of biochemistry.

[47]  D. Peabody,et al.  The RNA binding site of bacteriophage MS2 coat protein. , 1993, The EMBO journal.

[48]  B. Schnierle,et al.  The proline-rich region of the ecotropic Moloney murine leukaemia virus envelope protein tolerates the insertion of the green fluorescent protein and allows the generation of replication-competent virus. , 2003, The Journal of general virology.

[49]  S. Yu,et al.  Engineering the splice acceptor for improved gene expression and viral titer in an MLV-based retroviral vector , 2004, Gene therapy.

[50]  Qiang Zhou,et al.  Stimulatory effect of splicing factors on transcriptional elongation , 2001, Nature.

[51]  Peter J. Shepard,et al.  Conserved RNA secondary structures promote alternative splicing. , 2008, RNA.

[52]  D. Thomas,et al.  A Janus splicing regulatory element modulates HIV-1 tat and rev mRNA production by coordination of hnRNP A1 cooperative binding. , 2002, Journal of molecular biology.

[53]  Bosiljka Tasic,et al.  Alternative pre-mRNA splicing and proteome expansion in metazoans , 2002, Nature.

[54]  J. Kjems,et al.  The sequence complementarity between HIV-1 5' splice site SD4 and U1 snRNA determines the steady-state level of an unstable env pre-mRNA. , 2001, RNA.

[55]  K. Nandabalan,et al.  Mutations in U1 snRNA bypass the requirement for a cell type-specific RNA splicing factor , 1993, Cell.

[56]  Heiner Schaal,et al.  Extended base pair complementarity between U1 snRNA and the 5′ splice site does not inhibit splicing in higher eukaryotes, but rather increases 5′ splice site recognition , 2005, Nucleic acids research.

[57]  C. Stoltzfus,et al.  Role of viral splicing elements and cellular RNA binding proteins in regulation of HIV-1 alternative RNA splicing. , 2006, Current HIV research.

[58]  Rolf Backofen,et al.  Pre-mRNA Secondary Structures Influence Exon Recognition , 2007, PLoS genetics.

[59]  E. Buratti,et al.  Influence of RNA Secondary Structure on the Pre-mRNA Splicing Process , 2004, Molecular and Cellular Biology.

[60]  A. Schambach,et al.  New Way of Regulating Alternative Splicing in Retroviruses: the Promoter Makes a Difference , 2007, Journal of Virology.

[61]  A. Furger,et al.  Promoter proximal splice sites enhance transcription. , 2002, Genes & development.

[62]  D. Purcell,et al.  Alternative splicing of human immunodeficiency virus type 1 mRNA modulates viral protein expression, replication, and infectivity , 1993, Journal of virology.

[63]  Ravi Sachidanandam,et al.  Determinants of the inherent strength of human 5' splice sites. , 2005, RNA.

[64]  Jørgen Kjems,et al.  Defining a 5' splice site by functional selection in the presence and absence of U1 snRNA 5' end. , 2002, RNA.

[65]  C. Guthrie,et al.  An RNA switch at the 5' splice site requires ATP and the DEAD box protein Prp28p. , 1999, Molecular cell.

[66]  Robert H Singer,et al.  Structural basis for the coevolution of a viral RNA–protein complex , 2008, Nature Structural &Molecular Biology.

[67]  K. Beemon,et al.  Intronic sequences and 3' splice sites control Rous sarcoma virus RNA splicing , 1992, Journal of virology.

[68]  H. Kräusslich,et al.  Mutation of the major 5′ splice site renders a CMV‐driven HIV‐1 proviral clone Tat‐dependent: connections between transcription and splicing , 2004, FEBS letters.

[69]  J. Valcárcel,et al.  The hnRNP A1 protein regulates HIV‐1 tat splicing via a novel intron silencer element , 2001, The EMBO journal.

[70]  A. Mouland,et al.  The retrovirus RNA trafficking granule: from birth to maturity , 2006, Retrovirology.

[71]  J. Kjems,et al.  A 5' splice site enhances the recruitment of basal transcription initiation factors in vivo. , 2008, Molecules and Cells.

[72]  B. Ganem.  RNA world , 1987, Nature.

[73]  M. Zuker.  Computer prediction of RNA structure. , 1989, Methods in enzymology.

[74]  D. Zychlinski,et al.  Murine Leukemia Virus Regulates Alternative Splicing through Sequences Upstream of the 5· Splice Site* , 2006, Journal of Biological Chemistry.

[75]  K. Beemon,et al.  Two strong 5' splice sites and competing, suboptimal 3' splice sites involved in alternative splicing of human immunodeficiency virus type 1 RNA. , 1995, Virology.

[76]  E. Buratti,et al.  The Secondary Structure of the Human Immunodeficiency Virus Type 1 Transcript Modulates Viral Splicing and Infectivity , 2008, Journal of Virology.