The 3â² termini of small RNAs in<i>Trypanosoma brucei</i>

Hitchcock, Robert A.; Zeiner, Gusti M.; Sturm, Nancy R.; Campbell, David A.

doi:10.1111/j.1574-6968.2004.tb09629.x

Cited by 7 publications

(1 citation statement)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Ligation-mediated Terminal Analyses-U3 snoRNA 3Ј ends were determined by a three step ligation-mediated PCR amplification protocol essentially as described by Hitchcock et al (28). A 5Ј end phosphorylated (pCAGGAAACAGCUAUGAC) RNA adapter oligonucleotide (300 pmol) was first ligated to the 3Ј-hydroxyl groups total cellular RNA (1 g) using 10 units of T4 RNA ligase (Fermentas, Inc.) in 15 l of ligation buffer.…”

Section: Preparation and Analysis Of Cellular U3 Snornas Andmentioning

confidence: 99%

Nonpolyadenylated RNA Polymerase II Termination Is Induced by Transcript Cleavage

Nabavi

Nazar

2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

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 plasmidderived 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.In eukaryotic cells, the diversity in their DNA-dependent RNA polymerases appears also to be reflected in the mechanisms of transcription termination that each polymerase utilizes. Although studied most intensely, the mechanism(s) of Pol 2 II transcript termination is probably the least understood. In the case of pre-mRNA transcripts, 3Ј end processing and termination are normally coupled; the same RNA signals that direct 3Ј end processing also promote termination downstream of the processing site. At least two alternative models, which are not necessarily mutually exclusive, have been proposed (1). The first, often referred to as the "anti-terminator" model, suggests that polyadenylation triggers a change in polymerase-associated 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.In addition to mRNA, Pol II is responsible for the synthesis of many noncoding RNAs, including small nuclear and nucleolar RNAs (snRNA and snoRNA

show abstract

Section: Preparation and Analysis Of Cellular U3 Snornas Andmentioning

confidence: 99%

Nonpolyadenylated RNA Polymerase II Termination Is Induced by Transcript Cleavage

Nabavi

Nazar

2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

Fail-Safe Transcriptional Termination for Protein-Coding Genes in S. cerevisiae

et al. 2009

View full text Add to dashboard Cite

SummaryTranscription termination of RNA polymerase II (Pol II) on protein-coding genes in S. cerevisiae relies on pA site recognition by 3′ end processing factors. Here we demonstrate the existence of two alternative termination mechanisms that rescue polymerases failing to disengage from the template at pA sites. One of these fail-safe mechanisms is mediated by the NRD complex, similar to termination of short noncoding genes. The other termination mechanism is mediated by Rnt1 cleavage of the nascent transcript. Both fail-safe termination mechanisms trigger degradation of readthrough transcripts by the exosome. However, Rnt1-mediated termination can also enhance the usage of weak pA signals and thereby generate functional mRNA. We propose that these alternative Pol II termination pathways serve the dual function of avoiding transcription interference and promoting rapid removal of aberrant transcripts.

show abstract

Hypermethylated cap 4 maximizes Trypanosoma brucei translation

Zamudio

Mittra

Campbell

et al. 2009

Molecular Microbiology

View full text Add to dashboard Cite

SummaryThrough trans-splicing of a 39-nt spliced leader (SL) onto each protein-coding transcript, mature kinetoplastid mRNA acquire a hypermethylated 5Ј-cap structure, but its function has been unclear. Gene deletions for three Trypanosoma brucei cap 2Ј-Oribose methyltransferases, TbMTr1, TbMTr2 and TbMTr3, reveal distinct roles for four 2Ј-O-methylated nucleotides. Elimination of individual gene pairs yields viable cells; however, attempts at double knock-outs resulted in the generation of a TbMTr2 -/-/ TbMTr3 -/-cell line only. Absence of both kinetoplastid-specific enzymes in TbMTr2 -/-/TbMTr3 -/-lines yielded substrate SL RNA and mRNA with cap 1. TbMTr1 -/-translation is comparable with wildtype, while cap 3 and cap 4 loss reduced translation rates, exacerbated by the additional loss of cap 2. TbMTr1 -/-and TbMTr2 -/-/TbMTr3 -/-lines grow to lower densities under normal culture conditions relative to wildtype cells, with growth rate differences apparent under low serum conditions. Cell viability may not tolerate delays at both the nucleolar Sm-independent and nucleoplasmic Sm-dependent stages of SL RNA maturation combined with reduced rates of translation. A minimal level of mRNA cap ribose methylation is essential for trypanosome viability, providing the first functional role for the cap 4.

show abstract

The 3â² termini of small RNAs inTrypanosoma brucei

Cited by 7 publications

References 39 publications

Nonpolyadenylated RNA Polymerase II Termination Is Induced by Transcript Cleavage

Nonpolyadenylated RNA Polymerase II Termination Is Induced by Transcript Cleavage

Fail-Safe Transcriptional Termination for Protein-Coding Genes in S. cerevisiae

Hypermethylated cap 4 maximizes Trypanosoma brucei translation

Contact Info

Product

Resources

About

The 3â² termini of small RNAs inTrypanosoma brucei

Cited by 7 publications

References 39 publications

Nonpolyadenylated RNA Polymerase II Termination Is Induced by Transcript Cleavage

Nonpolyadenylated RNA Polymerase II Termination Is Induced by Transcript Cleavage

Fail-Safe Transcriptional Termination for Protein-Coding Genes in S. cerevisiae

Hypermethylated cap 4 maximizes Trypanosoma brucei translation

Contact Info

Product

Resources

About

The 3â² termini of small RNAs inTrypanosoma brucei