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Abstract
Although the termination of transcription and 3' RNA processing of the eukaryotic mRNA has been linked to a polyadenylation signal and a transcript cleavage process, much less is known about the termination or processing of nonpolyadenylated RNA polymerase II transcripts. An efficiently expressed plasmid-based expression system was used to study the termination and processing of Schizosaccharomyces pombe U3 small nucleolar RNA (snoRNA) transcripts in vivo. The termination assay was linked to cell transformation, and restriction fragment length polymorphism was used to determine levels of plasmid-derived U3 snoRNA. Mutation analyses in vivo indicate that the maturation of the 3' end is not directly dependent on an external cis-acting sequence or structure; rather, it is dependent on a transcript cleavage that can occur hundreds or even thousands of nucleotides downstream of the mature U3 snoRNA sequence. Similarly, termination is dependent on the same transcript cleavage that is localized in a hairpin structure that normally follows the 3' end of the U3 snoRNA but that also can be moved hundreds or thousands of nucleotides downstream. Both processes, however, can be induced simultaneously and equally efficiently with a single unrelated Pac1 endonuclease-labile structure. The results support a "reversed torpedoes" model in which a single cleavage allows exonucleases and/or other protein factors access to the transcript leading to transcription termination in one direction and RNA maturation in the other direction.
Highlights
MAY 16, 2008 VOLUME 283 NUMBER 20 ated factors that leads to a less processive RNA polymerase; an alternate model suggests that the transcribing RNA polymerase is “torpedoed,” being attacked by an exonuclease that gains access at its free end when the pre-mRNA is cut at the polyadenylation site
Numerous other protein factors have been implicated in nonpolyadenylated Pol II termination including a helicase (Sen1) and subunits of the APT complex (Pti1, Ref2, Ssu72, and Swd2), which associate with mRNA cleavage/polyadenylation factor Pta1 (8 –13)
An efficiently expressed plasmid associated gene system was used to study the expression and maturation of U3 small nucleolar RNA (snoRNA) encoded by the S. pombe snU32 locus
Summary
Strains and Vectors—Escherichia coli strain C490 (recAϪ, rkϪ, mkϪ, thrϪ, leuϪ, metϪ) was used as a host for pTZ19R [16] and pFL20 [17] plasmid recombinants. When appropriate, primers with BamHI adapter sequences at their ends were used to prepare the final fragments; after digestion with BamHI endonuclease, the mutant gene sequences were cloned directly into the pFL20 yeast shuttle vector using its BamHI site. U3 cDNA was prepared from the gel-purified RNA by RT-PCR [27] using Moloney murine leukemia virus reverse transcriptase and primers specific for the 5Ј and 3Ј ends of the S. pombe U3 snoRNAs (5Ј-ATCGACGATACTCCATAG-3Ј and 5Ј-ACACGTCAGAAAACACC-3Ј), respectively, as described above. The resulting cDNA solution was diluted to 20 l, and a 2-l aliquot was used for PCR amplification, with 30 pmol of complementary primer, 30 pmol of U3 snoRNA-specific primer (5Ј-ATCGACGATACTCCATAG-3Ј), and 2.5 units Taq DNA polymerase in 50 l of buffer (500 mM KCl, 15 mM MgCl2, 100 mM Tris-HCl, pH 9.0 containing 1% Triton X-100)
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