Specificity of RNA maturation pathways: RNAs transcribed by RNA polymerase III are not substrates for splicing or polyadenylation

To analyze the specificity of RNA processing reactions, we constructed hybrid genes containing RNA polymerase III promoters fused to sequences that are normally transcribed by polymerase II and assessed their transcripts following transfection into human 293 cells. Transcripts derived from these chimeric constructs were analyzed by using a combined RNase H and S1 nuclease assay to test whether RNAs containing consensus 5' and 3' splicing signals could be efficiently spliced in intact cells, even though they were transcribed by RNA polymerase III. We found that polymerase III-derived RNAs are not substrates for splicing. Similarly, we were not able to detect poly(A)+ RNAs derived from genes that contained a polymerase III promoter linked to sequences that were necessary and sufficient to direct 3'-end cleavage and polyadenylation when transcribed by RNA polymerase II. Our findings are consistent with the view that in vivo splicing and polyadenylation pathways are obligatorily coupled to transcription by RNA polymerase II.

[1]  D. Cleveland,et al.  A combination of RNase H and S1 nuclease circumvents an artefact inherent to conventional S1 analysis of RNA splicing. , 1987, Nucleic acids research.

[2]  Tom Maniatis,et al.  The role of small nuclear ribonucleoprotein particles in pre-mRNA splicing , 1987, Nature.

[3]  V. Natarajan.  Adenovirus-2 E1a and E1b gene products regulate enhancer mediated transcription. , 1986, Nucleic acids research.

[4]  J. Manley,et al.  Polyadenylylation of an mRNA precursor occurs independently of transcription by RNA polymerase II in vivo. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[5]  D. Cleveland,et al.  RNA polymerase specificity of mRNA production and enhancer action. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[6]  J. Nevins,et al.  Poly(A) site cleavage in a HeLa nuclear extract is dependent on downstream sequences , 1985, Cell.

[7]  Y. Osheim,et al.  RNP particles at splice junction sequences on Drosophila chorion transcripts , 1985, Cell.

[8]  N. Sonenberg,et al.  Cap-dependent RNA splicing in a HeLa nuclear extract. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[9]  D. Cleveland,et al.  Surprising S1-resistant trimolecular hybrids: potential complication in interpretation of S1 mapping analyses , 1985, Molecular and cellular biology.

[10]  T. Gurney,et al.  Characterization of mouse 45S ribosomal RNA subspecies suggests that the first processing cleavage occurs 600 +/- 100 nucleotides from the 5' end and the second 500 +/- 100 nucleotides from the 3' end of a 13.9 kb precursor , 1985, Nucleic Acids Res..

[11]  P. Sharp,et al.  Accurate cleavage and polyadenylation of exogenous RNA substrate , 1985, Cell.

[12]  P. Sharp,et al.  Splicing messenger RNA precursors: branch sites and lariat RNAs , 1985 .

[13]  J. McLauchlan,et al.  The consensus sequence YGTGTTYY located downstream from the AATAAA signal is required for efficient formation of mRNA 3' termini. , 1985, Nucleic acids research.

[14]  R. Tjian,et al.  Transcription of herpes simplex virus tk sequences under the control of wild-type and mutant human RNA polymerase I promoters , 1985, Molecular and cellular biology.

[15]  P. Sharp,et al.  Splicing of messenger RNA precursors. , 1985, Harvey lectures.

[16]  P. Sharp,et al.  Recognition of cap structure in splicing in vitro of mRNA precursors , 1984, Cell.

[17]  B Sollner-Webb,et al.  High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment. , 1984, Nucleic acids research.

[18]  Michael R. Green,et al.  Normal and mutant human β-globin pre-mRNAs are faithfully and efficiently spliced in vitro , 1984, Cell.

[19]  N. Proudfoot.  The end of the message and beyond. , 1984, Nature.

[20]  K. Miller,et al.  Dinucleotide primers facilitate convenient identification of the mouse ribosomal DNA transcription initiation site. A general method for analysis of transcription by RNA polymerases I and III. , 1983, The Journal of biological chemistry.

[21]  A. Lassar,et al.  Transcription of class III genes: formation of preinitiation complexes. , 1983, Science.

[22]  J. Darnell.  The processing of RNA. , 1983, Scientific American.

[23]  L. Chow,et al.  Four unique genes required for β tubulin expression in vertebrates , 1983, Cell.

[24]  Michael R. Green,et al.  Human β-globin pre-mRNA synthesized in vitro is accurately spliced in xenopus oocyte nuclei , 1983, Cell.

[25]  J. Abelson,et al.  Mechanism of action of a yeast RNA ligase in tRNA splicing , 1983, Cell.

[26]  J. Abelson,et al.  Precise excision of intervening sequences from precursor tRNAs by a membrane-associated yeast endonuclease , 1983, Cell.

[27]  Stephen M. Mount,et al.  A catalogue of splice junction sequences. , 1982, Nucleic acids research.

[28]  L. J. Korn.  Transcription of Xenopus 5S ribosomal RNA genes , 1982, Nature.

[29]  K. Miller,et al.  Transcription of mouse rRNA genes by RNA polymerase I: In vitro and in vivo initiation and processing sites , 1981, Cell.

[30]  J. Nevins,et al.  Nuclear RNA is spliced in the absence of poly(A) addition , 1981, Cell.

[31]  R. Axel,et al.  Analysis of transcriptional regulatory signals of the HSV thymidine kinase gene: Identification of an upstream control region , 1981, Cell.

[32]  D. Bogenhagen,et al.  Nucleotide sequences in Xenopus 5S DNA required for transcription termination , 1981, Cell.

[33]  T. Shenk,et al.  The sequence 5′-AAUAAA-3′ forms part of the recognition site for polyadenylation of late SV40 mRNAs , 1981, Cell.

[34]  R. Roeder,et al.  Multiple factors are required for the accurate transcription of purified genes by RNA polymerase III. , 1980, The Journal of biological chemistry.

[35]  J. Nevins,et al.  Addition of poly(A) to nuclear RNA occurs soon after RNA synthesis , 1980, The Journal of cell biology.

[36]  A. Banerjee,et al.  5'-terminal cap structure in eucaryotic messenger ribonucleic acids , 1980 .

[37]  D. Bogenhagen,et al.  A control region in the center of the 5S RNA gene directs specific initiation of transcription: I. The 5′ border of the region , 1980, Cell.

[38]  D. Bogenhagen,et al.  A control region in the center of the 5S RNA gene directs specific initiation of transcription: II. The 3′ border of the region , 1980, Cell.

[39]  J. Darnell,et al.  The addition of 5′ cap structures occurs early in hnRNA synthesis and prematurely terminated molecules are capped , 1980, Cell.

[40]  R. Treisman,et al.  Transcription maps of polyoma virus-specific RNA: analysis by two-dimensional nuclease S1 gel mapping. , 1980, Methods in enzymology.

[41]  A. K. Banerjee.  5'-terminal cap structure in eucaryotic messenger ribonucleic acids. , 1980, Microbiological reviews.

[42]  J. Nevins,et al.  N-6-methyl-adenosine in adenovirus type 2 nuclear RNA is conserved in the formation of messenger RNA. , 1979, Journal of molecular biology.

[43]  W. Rutter,et al.  Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. , 1979, Biochemistry.

[44]  Helen Donis-Keller,et al.  Site specific enzymatic cleavage of RNA , 1979, Nucleic Acids Res..

[45]  J. Darnell,et al.  Transcription units for mRNA production in eukaryotic cells and their DNA viruses. , 1979, Progress in nucleic acid research and molecular biology.

[46]  J. Abelson.  RNA processing and the intervening sequence problem. , 1979, Annual review of biochemistry.

[47]  W. Filipowicz.  Functions of the 5′‐terminal m7G cap in eukaryotic mRNA , 1978, FEBS letters.

[48]  P. Sharp,et al.  Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids , 1977, Cell.

[49]  N. Proudfoot,et al.  3′ Non-coding region sequences in eukaryotic messenger RNA , 1976, Nature.

[50]  R. Perry,et al.  Kinetics of formation of 5′ terminal caps in mRNA , 1976, Cell.

[51]  A. van der Eb,et al.  A new technique for the assay of infectivity of human adenovirus 5 DNA. , 1973, Virology.

[52]  R. Perry,et al.  Inhibition of RNA synthesis by actinomycin D: Characteristic dose‐response of different RNA species , 1970, Journal of cellular physiology.

[53]  E. Reich,et al.  Actinomycin and nucleic acid function. , 1964, Progress in nucleic acid research and molecular biology.