The 3? termini of small RNAs in Trypanosoma brucei

Hitchcock, Robert A.

doi:10.1016/j.femsle.2004.05.020

Cited by 8 publications

(10 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Recombinant SNIP had an in vitro preference for the exonucleolytic removal of U residues commonly found at the 3Ј ends of small RNAs and minimal activity on G or C ribonucleotides. Substrates for SNIP in vivo have G or C at their mature 3Ј ends, whereas nonsubstrates have U or A residues at the mature 3Ј ends (6). SNIP is an essential protein in T. brucei.…”

Section: Discussionmentioning

confidence: 99%

“…The mature 3Ј end of T. brucei SL RNA is C 142 (6). The next ribonucleotide downstream of the mature SL RNA 3Ј end is U 143 .…”

Section: Discussionmentioning

confidence: 99%

“…The 3Ј ends of SL RNA from mock-induced and TET-induced SNIP RNAi cells were mapped. The majority of 3Ј ends for SL RNA from uninduced cells was C 142 (6), whereas the majority of 3Ј ends from TETinduced cells was U 143 (data not shown). In the case of all but the 5S rRNA, which accumulated a 4-to 5-nt longer form, the substrate RNAs accumulated species 1 to 3 nt longer than their mature forms.…”

Section: Cloning Of T Brucei Snipmentioning

confidence: 92%

See 2 more Smart Citations

3′-End Polishing of the Kinetoplastid Spliced Leader RNA Is Performed by SNIP, a 3′→5′ Exonuclease with a Motley Assortment of Small RNA Substrates

Zeiner

Hitchcock

Sturm

et al. 2004

Molecular and Cellular Biology

View full text Add to dashboard Cite

In all trypanosomatids, trans splicing of the spliced leader (SL) RNA is a required step in the maturation of all nucleus-derived mRNAs. The SL RNA is transcribed with an oligo-U 3 extension that is removed prior to trans splicing. Here we report the identification and characterization of a nonexosomal, 335 exonuclease required for SL RNA 3-end formation in Trypanosoma brucei. We named this enzyme SNIP (for snRNA incomplete 3 processing). The central 158-amino-acid domain of SNIP is related to the exonuclease III (ExoIII) domain of the 335 proofreading subunit of Escherichia coli DNA polymerase III holoenzyme. SNIP had a preference for oligo(U) 3 extensions in vitro. RNA interference-mediated knockdown of SNIP resulted in a growth defect and correlated with the accumulation of one-to two-nucleotide 3 extensions of SL RNA, U2 and U4 snRNAs, a five-nucleotide extension of 5S rRNA, and the destabilization of U3 snoRNA and U2 snRNA. SNIP-green fluorescent protein localized to the nucleoplasm, and substrate SL RNA derived from SNIP knockdown cells showed wild-type cap 4 modification, indicating that SNIP acts on SL RNA after cytosolic trafficking. Since the primary SL RNA transcript was not the accumulating species in SNIP knockdown cells, SL RNA 3-end formation is a multistep process in which SNIP provides the ultimate 3-end polishing. We speculate that SNIP is part of an organized nucleoplasmic machinery responsible for processing of SL RNA.The kinetoplastid protozoa are deep-branching eukaryotes that have evolved unusual mechanisms of nuclear gene expression. Transcription initiation of protein-coding genes in these organisms is regulated minimally, and all nucleus-derived premRNAs are transcribed polycistronically. To resolve these polycistronic pre-mRNAs into mature mRNAs, kinetoplastids use the trans-splicing reaction. Through trans splicing, the 5Ј 39-nucleotide (nt) spliced leader (SL) is transferred from the substrate SL RNA to the polycistronic pre-mRNA, yielding transspliced mRNAs. In addition to substrate SL RNA, trans splicing also requires the coordinated functions of mature U2, U4, U5, and U6 small nuclear RNAs (snRNAs) and associated proteins.Although there are differences in the details of individual snRNA gene transcription among eukaryotes, all snRNAs share at least one common feature: nascent snRNAs contain 3Ј extensions as a consequence of transcription termination. These 3Ј extensions are of variable sequence and length and are removed to yield the mature 3Ј end of each snRNA (10). In metazoans 3Ј-extension removal of nascent U1 snRNA was shown to require (i) intracellular trafficking of U1 snRNA, (ii) the activities of at least two nucleases, and (iii) a cis-acting element downstream of the mature 3Ј end of U1 snRNA (5). The first nuclease acting on nascent U1 snRNA was cytosolic and left a 2-nt extension, whereas the final 3Ј-end trimming activity was nuclear (20). 3Ј-Extension removal of human precursor U2 snRNA also required intracellular trafficking, a 3Ј cis-acting sequence element, and at...

show abstract

Section: Discussionmentioning

confidence: 99%

“…The mature 3Ј end of T. brucei SL RNA is C 142 (6). The next ribonucleotide downstream of the mature SL RNA 3Ј end is U 143 .…”

Section: Discussionmentioning

confidence: 99%

Section: Cloning Of T Brucei Snipmentioning

confidence: 92%

See 1 more Smart Citation

3′-End Polishing of the Kinetoplastid Spliced Leader RNA Is Performed by SNIP, a 3′→5′ Exonuclease with a Motley Assortment of Small RNA Substrates

Zeiner

Hitchcock

Sturm

et al. 2004

Molecular and Cellular Biology

View full text Add to dashboard Cite

show abstract

“…LM-RT-PCR was performed as described (Hitchcock et al., 2004) with modifications presented in the Supplemental Data.…”

Section: Methodsmentioning

confidence: 99%

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

“…In wild-type cells SL RNA was present in three main forms: a 141-nt species, the mature 142-nt molecule (20), and the 143-nt TbSNIP substrate, representing a 71:29 ratio of mature to underprocessed SL RNA. TbMTr1-null lines showed a decrease in mature 142-nt and accumulation of 144-to 150-nt forms, representing a 21:79 ratio of mature to underprocessed SL RNA (Fig.…”

Section: Tbmt370 and Conserved Viral And Eukaryotic 2-o-ribosementioning

confidence: 99%

The 2′-O-Ribose Methyltransferase for Cap 1 of Spliced Leader RNA and U1 Small Nuclear RNA inTrypanosoma brucei

Zamudio

Mittra

Foldynová-Trantírková

et al. 2007

Molecular and Cellular Biology

View full text Add to dashboard Cite

The m 7 G cap on eukaryotic mRNA plays essential roles in translation and stability, and the enzymatic activities responsible for its formation are well characterized (48, 50). Additional modifications of adjacent nucleotides on mRNA and small nuclear RNA (snRNA) are well documented; however, the identity of the eukaryotic cap ribose-methylating enzyme(s) and the function of cap modification beyond the m 7 G attached to the first-transcribed nucleotide (cap 0) remain unclear (6, 18). 2Ј-O-Ribose methylation is a common modification of RNA (29) that can occur via two mechanisms: box C/D snoRNA-guided methylation by fibrillarin (12) and nucleotidespecific enzymes (31,43,54). The conservation of modified nucleotides in functionally important regions indicates a major benefit to the cell (13). Individual methylations of tRNA are not essential for viability; however, their cumulative loss is detrimental (1). Potential functions for mRNA 2Ј-O-ribose methylation include roles in processing and trafficking of transcripts, as well as translational control (4, 45).Protein-encoding genes of kinetoplastid protozoa, including the human-pathogenic Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi, are transcribed polycistronically (23,36). The discrete spliced leader (SL) RNA serves as donor of its first 39 nucleotides (nt) and unique cap structure to all coding regions via trans-splicing. In addition to m 7 G, the hypermethylated cap 4 structure includes 2Ј-O-ribose methylations of the first 4 nt (AACU) with additional base methylations on the first (m 2 6 A) and fourth (m 3 U) positions (5, 16, 41). The 5Ј processing of SL is accompanied by association of the transcript with an Sm complex between two stem-loop structures in the intron (35,51,65) and 3Ј trimming of residual nucleotides from attenuated transcription termination in the downstream T tract (51, 66). Other eukaryotic mRNA caps vary from those containing only cap 0 to those modified by 2Ј-O-ribose methylation(s) at the first or the first and second nucleotides, known as cap 1 and cap 2, respectively (17). Among higher eukaryotes cap ribose modifications are conserved, and the majority of mRNAs are ribose methylated in mammals (4).The unique kinetoplastid cap 4 structure is implicated both in trans-splicing and in translation. Substrate SLs with incomplete cap 4 are trans spliced (52); however, these mRNAs do not associate with polysomes (68), suggesting a role for cap 4, the primary SL sequence, or both in translation. Two cap 2Ј-O-ribose methyltransferases (MTases), TbMT417 and TbMT511, that modify the second and third positions of cap 4, are dispensable for survival of T. brucei (2,3,64). These relatives of the vaccinia virus cap 1 MTase will henceforth be referred to as TbMTr2 and TMTr3, respectively, to reflect their functional roles. With no requirement for TbMTr2 and TbMTr3 activities for viability, the focus for a critical component of cap 4 turned to position 1.

show abstract

The 3? termini of small RNAs in Trypanosoma brucei

Cited by 8 publications

References 34 publications

3′-End Polishing of the Kinetoplastid Spliced Leader RNA Is Performed by SNIP, a 3′→5′ Exonuclease with a Motley Assortment of Small RNA Substrates

3′-End Polishing of the Kinetoplastid Spliced Leader RNA Is Performed by SNIP, a 3′→5′ Exonuclease with a Motley Assortment of Small RNA Substrates

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

The 2′-O-Ribose Methyltransferase for Cap 1 of Spliced Leader RNA and U1 Small Nuclear RNA inTrypanosoma brucei

Contact Info

Product

Resources

About