Characterization of the structure, function, and mechanism of B2 RNA, an ncRNA repressor of RNA polymerase II transcription.

We previously found that the SINE-encoded mouse B2 RNA binds RNA polymerase II and represses mRNA transcription during the cellular heat-shock response. In vitro B2 RNA assembles into preinitiation complexes on promoter DNA via its interaction with the polymerase, thus rendering the complexes inactive. With the goal of understanding which regions of B2 RNA are important for high-affinity binding to RNA polymerase II and repression of transcription, we performed a structural and deletion analysis of a 178 nucleotide (nt) B2 RNA. We describe an experimentally derived secondary structure model for B2 RNA, and show that RNA polymerase II protects a specific region from RNase digestion. Deletion studies revealed that a 51-nt region of B2 RNA is sufficient for high-affinity binding to RNA polymerase II, association with preinitiation complexes, and repression of transcription in vitro, the latter of which involves a large predominately single-stranded region within the RNA. In addition, this piece of B2 RNA blocked the polymerase from properly associating with template DNA during the assembly of elongation complexes. Further deletion revealed that a 33-nt piece of B2 RNA binds RNA polymerase II, associates with preinitiation complexes, but cannot repress transcription. These results support a model in which RNA polymerase II contains a high-affinity ncRNA docking site to which a distinct region of B2 RNA binds, thereby allowing another region of the RNA to repress transcription. Moreover, the mechanism of transcriptional repression by B2 RNA likely involves disrupting critical contacts between RNA polymerase II and the promoter DNA.

[1]  S. Gaffin,et al.  Effect of acute heat shock on gene expression by human peripheral blood mononuclear cells. , 2002, Journal of applied physiology.

[2]  R. Kingston,et al.  Cooperation between Complexes that Regulate Chromatin Structure and Transcription , 2002, Cell.

[3]  R. Tjian,et al.  Transcription factors IIE and IIH and ATP hydrolysis direct promoter clearance by RNA polymerase II , 1994, Cell.

[4]  J. Lis,et al.  Rapid changes in Drosophila transcription after an instantaneous heat shock , 1993, Molecular and cellular biology.

[5]  J. Goodrich,et al.  In vitro studies of the early steps of RNA synthesis by human RNA polymerase II. , 2003, Methods in enzymology.

[6]  J. Sabina,et al.  Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. , 1999, Journal of molecular biology.

[7]  P. Rigby,et al.  Regulation of RNA polymerase III transcription in response to F9 embryonal carcinoma stem cell differentiation , 1989, Cell.

[8]  R R Breaker,et al.  Relationship between internucleotide linkage geometry and the stability of RNA. , 1999, RNA.

[9]  J. Goodrich,et al.  The SINE-encoded mouse B2 RNA represses mRNA transcription in response to heat shock , 2004, Nature Structural &Molecular Biology.

[10]  J. Goodrich,et al.  Translocation after Synthesis of a Four-Nucleotide RNA Commits RNA Polymerase II to Promoter Escape , 2002, Molecular and Cellular Biology.

[11]  C. Carles,et al.  Selective Targeting and Inhibition of Yeast RNA Polymerase II by RNA Aptamers* , 1997, The Journal of Biological Chemistry.

[12]  C. Rudin,et al.  Transcriptional activation of short interspersed elements by DNA‐damaging agents , 2001, Genes, chromosomes & cancer.

[13]  R. Tjian,et al.  Transcriptional coactivator complexes. , 2001, Annual review of biochemistry.

[14]  A. Fornace,et al.  Induction of B2 RNA polymerase III transcription by heat shock: enrichment for heat shock induced sequences in rodent cells by hybridization subtraction. , 1986, Nucleic acids research.

[15]  R. Bachvarova.  Small B2 RNAs in mouse oocytes, embryos, and somatic tissues. , 1988, Developmental biology.

[16]  P. Cramer,et al.  Structure of an RNA polymerase II–RNA inhibitor complex elucidates transcription regulation by noncoding RNAs , 2006, Nature Structural &Molecular Biology.

[17]  J. Goodrich,et al.  The Sequence at Specific Positions in the Early Transcribed Region Sets the Rate of Transcript Synthesis by RNA Polymerase II in Vitro* , 2005, Journal of Biological Chemistry.

[18]  T Lagrange,et al.  The general transcription factors of RNA polymerase II. , 1996, Genes & development.

[19]  M. Kashlev,et al.  The 8-Nucleotide-long RNA:DNA Hybrid Is a Primary Stability Determinant of the RNA Polymerase II Elongation Complex* , 2000, The Journal of Biological Chemistry.

[20]  J. Lis,et al.  In vivo interactions of RNA polymerase II with genes of Drosophila melanogaster , 1985, Molecular and cellular biology.

[21]  G. Storz,et al.  An abundance of RNA regulators. , 2005, Annual review of biochemistry.

[22]  T. S. Brown,et al.  Method for assigning double-stranded RNA structures. , 2005, BioTechniques.

[23]  W. L. Ruzzo,et al.  6S RNA is a widespread regulator of eubacterial RNA polymerase that resembles an open promoter. , 2005, RNA.

[24]  R. Roeder,et al.  Transcriptional regulation through Mediator-like coactivators in yeast and metazoan cells. , 2000, Trends in biochemical sciences.

[25]  K. Wassarman,et al.  A highly conserved 6S RNA structure is required for regulation of transcription , 2005, Nature Structural &Molecular Biology.

[26]  T. Pederson,et al.  Regulated transcription of the genes for actin and heat-shock proteins in culture drosophila cells , 1981, The Journal of cell biology.

[27]  Celso A. Espinoza,et al.  B2 RNA binds directly to RNA polymerase II to repress transcript synthesis , 2004, Nature Structural &Molecular Biology.

[28]  C. Schmid,et al.  Cell stress and translational inhibitors transiently increase the abundance of mammalian SINE transcripts. , 1995, Nucleic acids research.

[29]  C. Schmid,et al.  Physiological stresses increase mouse short interspersed element (SINE) RNA expression in vivo. , 1999, Gene.

[30]  K. Wassarman,et al.  Synthesis-Mediated Release of a Small RNA Inhibitor of RNA Polymerase , 2006, Science.

[31]  J. Goodrich,et al.  Non-coding-RNA regulators of RNA polymerase II transcription , 2006, Nature Reviews Molecular Cell Biology.

[32]  Craig M. Lilly,et al.  Selected Contribution: Effect of acute heat shock on gene expression by human peripheral blood mononuclear cells , 2002 .

[33]  M. Botchan,et al.  Expression of enhanced levels of small RNA polymerase III transcripts encoded by the B2 repeats in simian virus 40-transformed mouse cells , 1985, Nature.

[34]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[35]  Dimitri A Kramerov,et al.  Short retroposons in eukaryotic genomes. , 2005, International review of cytology.

[36]  G. Storz,et al.  6S RNA Regulates E. coli RNA Polymerase Activity , 2000, Cell.

[37]  S. Tillib,et al.  The most abundant nascent poly(A) + RNAs are transcribed by RNA polymerase III in murine tumor cells. , 1990, Nucleic acids research.