Characterization of the RNA polymerases of Trypanosoma brucei: trypanosomal mRNAs are composed of transcripts derived from both RNA polymerase II and III.

Grondal, Ernst J.M.; Evers, Raymond; Kosubek, K.; Cornelissen, A. W. C. A.

doi:10.1002/j.1460-2075.1989.tb08502.x

Cited by 71 publications

(59 citation statements)

References 48 publications

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“…AGTC (41,64). This observation has led to suggestions that VSG genes and PARP genes are transcribed either in the nucleolus along with the rRNA genes (59,75) or by a modified RNA polymerase 11 (26,32). Recently, the promoter for the PARP genes was shown by deletion analyses and linker scanning mutagenesis to encompass about 230 bp immediately upstream of the transcription start site, including a core region and an upstream control element (7,64).…”

Section: Discussionmentioning

confidence: 99%

“…Nuclear run-on assays, coupled with UV inactivation of transcription, have been used to demonstrate that for at least some VSG ESs, transcription is initiated 45 to 60 kb upstream of the VSG gene and proceeds through eight or more open translation reading frames, the last of which specifies the VSG (12,30,37,42,56). This transcription occurs in the presence of ox-amanitin, leading to suggestions that it is catalyzed by an RNA polymerase I activity or by a modified RNA polymerase II complex (26,32,59,75). The resultant polycistronic transcript is processed into the individual mRNAs by 5' trans splicing and 3' polyadenylation.…”

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confidence: 99%

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A monocistronic transcript for a trypanosome variant surface glycoprotein.

Alarcon¹,

Son²,

Hall³

et al. 1994

Mol. Cell. Biol.

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Many protein-encoding genes of African trypanosomes are transcribed as large polycistronic pre-mRNAs that are processed into individual mRNAs containing a 5' spliced leader and 3' poly(A). The 45-to 60-kb pre-mRNAs encoding some variant surface glycoproteins (VSGs) contain as many as eight unrelated coding regions. Here we identify the promoter for a metacyclic VSG gene that is expressed without duplication in a bloodstream trypanosome clone. This 70-bp promoter is located 2 kb upstream of the telomere-linked VSG gene and directs the synthesis of a monocistronic VSG pre-mRNA lacking the 5' spliced leader. Its sequence only slightly resembles those of other known trypanosome promoters, but it does cross-hybridize with several related sequences elsewhere in the genome. These results suggest that a new class of trypanosome promoters has been found, whose function is to initiate monocistronic transcription of those VSG genes normally expressed during the metacyclic stage.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

A monocistronic transcript for a trypanosome variant surface glycoprotein.

Alarcon¹,

Son²,

Hall³

et al. 1994

Mol. Cell. Biol.

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“…The presence of all three RNAPs in Trypanosoma brucei has been demonstrated by Mono-Q anion exchange chromatography and nuclear run-on experiments with polymerase inhibitors (Grondal et al, 1989), while in Leishmania, they have been separated by a combination of carboxymethyl-sephadex and diethylaminoethyl-sephadex chromatography (Sadhukhan et al, 1997). BLAST analysis of the recently sequenced Leishmania major, T. brucei and Trypanosoma cruzi (Tritryp) genomes (Ivens et al, 2005) revealed the presence of all the shared and homologous subunits, with the exception of RPB12, RPA43 and RPA14/RPB4/RPC9, but most of the RNAP-specific subunits were not identified.…”

Section: Introductionmentioning

confidence: 99%

Characterization of the RNA polymerase II and III complexes in Leishmania major

Martínez‐Calvillo

Saxena

Green

et al. 2007

International Journal for Parasitology

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Transcription of protein-coding genes in Leishmania major and other trypanosomatids differs from that in most eukaryotes and bioinformatic analyses have failed to identify several components of the RNA polymerase (RNAP) complexes. To increase our knowledge about this basic cellular process, we used tandem affinity purification (TAP) to identify subunits of RNAP II and III. Mass spectrometric analysis of the complexes co-purified with TAP-tagged LmRPB2 (encoded by LmjF31.0160) identified seven RNAP II subunits: RPB1, RPB2, RPB3, RPB5, RPB7, RPB10 and RPB11. With the exception of RPB10 and RPB11, and the addition of RPB8, these were also identified using TAP-tagged constructs of one (encoded by LmjF34.0890) of the two LmRPB6 orthologues. The latter experiments also identified the RNAP III subunits RPC1 (C160), RPC2 (C128), RPC3 (C82), RPC4 (C53), RPC5 (C37), RPC6 (C34), RPC9 (C17), RPAC1 (AC40) and RPAC2 (AC19). Significantly, the complexes precipitated by TAP-tagged LmRPB6 did not contain any RNAP I-specific subunits, suggesting that, unlike in other eukaryotes, LmRPB6 is not shared by all three polymerases but is restricted to RNAP II and III, while the LmRPB6z (encoded by LmjF25.0140) isoform is limited to RNAP I. Similarly, we identified peptides from only one (encoded by LmjF18.0780) of the two RPB5 orthologues and one (LmjF13.1120) of the two RPB10 orthologues, suggesting that LmRPB5z (LmjF18.0790) and LmRPB10z (LmjF13.1120) are also restricted to RNAP I. In addition to these RNAP subunits, we also identified a number of other proteins that co-purified with the RNAP II and III complexes, including a potential transcription factor, several histones, an ATPase involved in chromosome segregation, an endonuclease, four helicases, RNA splicing factor PTSR-1, at least two RNA binding proteins and several proteins of unknown function.

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“…Purified GTases isolated from other sources cap 5'-tri-or -diphosphate RNAs indiscriminately, and the specificity of the capping reaction for Pol II transcripts in vivo is thought to reside within the Pol II transcription initiation complex (6). However, since it has been argued that the SL-RNA is transcribed by Pol III (10), it is unlikely that the capping activity in the kinetoplastid protozoa is limited to the Pol II initiation complex. It is particularly interesting that the apparent specificity of the in vitro T. cruzi activity closely reflects the in vivo capping requirements of the kinetoplastid protozoa.…”

Section: Resultsmentioning

confidence: 99%

“…refs. 7 and 8), and some protein coding genes may not be transcribed by a normal a-amanitin-sensitive Pol 11 (9)(10)(11). Thus, it has been proposed that trans-splicing evolved in the kinetoplastid protozoa as a means of capping protein coding mRNAs (12).…”

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confidence: 99%

RNA-protein complexes mediate in vitro capping of the spliced-leader primary transcript and U-RNAs in Trypanosoma cruzi.

Zwierzynski¹,

Buck²

1991

Proc. Natl. Acad. Sci. U.S.A.

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A 39-nucleotide spliced leader (SL) is joined to the 5' ends of trypanosome mRNAs in a bimolecular or trans-splicing process. The SL in Trypanosoma cruzi is transcribed as an lO-nucleotide RNA (SL-RNA or SL primary transcript) bearing the 39-nucleotide SL at the 5' end. The SL-RNA is 5' capped by a guanylyltransferase activity prior to trans-splicing and trypanosome mRNAs thus obtain their mature caps from the SL by trans-splicing. We have previously characterized a guanylyltransferase activity from T. cruzi nuclear extracts and shown that this capping activity has an unusual ATP dependence and an apparent specificity for the SL-RNA and U-RNAs. Herein, we show that the capping activity sediments as a 12-15S particle during velocity sedimentation in glycerol gradients and fractionates as a >150-kDa particle during large-pore gel filtration chromatography. Moreover, the endogenous substrate RNAs-the SL-RNA and U-RNAs-consistently copurify with the capping activity, suggesting that the activity and the substrates form a ribonucleoprotein particle. The capping activity and substrate RNAs are not dissociated in isopycnic Cs2SO4 gradients and band at a density expected for an RNA-protein complex, confirming the existence of ribonucleoprotein particles bearing both the activity and its substrate RNAs. Finally, we partially purified these ribonucleoprotein particles and showed that the capping activity remains ATP dependent and highly specific for the SL-RNA and the U-RNAs. These observations are consistent with the hypothesis that one of the functions of trans-splicing is for mRNA capping. mRNA maturation in the kinetoplastid protozoa requires an unusual trans-splicing process in which a 39-nucleotide leader, the spliced leader (SL), from a small abundant transcript, the SL primary transcript (SL-RNA), is ligated to the 5' ends of structural gene primary transcripts (1). The SL-RNA obtains an unusual 5' cap that is transferred to the mature mRNA with the SL (2-5). In other eukaryotes, mRNA primary transcripts are capped shortly after transcription initiation by guanylyltransferases (GTases) that are probably associated with the RNA polymerase II (Pol II) initiation complex in a reaction requiring a 5'-tri-or -diphosphate substrate RNA (6). Although capping is thought to be mediated by the Pol II transcription initiation complex (6), soluble partially purified GTases lack substrate specificity and require only the energy from cleavage of the GTP substrate for activity. In the kinetoplastid protozoa, there is no evidence that mRNA primary transcripts are capped, many genes are transcribed as "multicistronic" RNAs that are processed into monocistronic mRNAs (cf. refs. 7 and 8), and some protein coding genes may not be transcribed by a normal a-amanitin-sensitive Pol 11 (9-11). Thus, it has been proposed that trans-splicing evolved in the kinetoplastid protozoa as a means of capping protein coding mRNAs (12).We have established (13) nuclear extracts from Trypanosoma cruzi that cap the SL-RNA and at least two U-RNAs...

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Characterization of the RNA polymerases of Trypanosoma brucei: trypanosomal mRNAs are composed of transcripts derived from both RNA polymerase II and III.

Cited by 71 publications

References 48 publications

A monocistronic transcript for a trypanosome variant surface glycoprotein.

A monocistronic transcript for a trypanosome variant surface glycoprotein.

Characterization of the RNA polymerase II and III complexes in Leishmania major

RNA-protein complexes mediate in vitro capping of the spliced-leader primary transcript and U-RNAs in Trypanosoma cruzi.

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