Accurate Transcription of the Trypanosoma brucei U2 Small Nuclear RNA Gene in a Homologous Extract

Günzl, Arthur; Tschudi, Christian; Nakaar, Valerian; Ullu, Elisabetta

doi:10.1074/jbc.270.29.17287

Cited by 26 publications

(25 citation statements)

References 18 publications

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“…Labeled SL RNA was produced from wild-type using the permeable cell system, as described above . Whole cell extracts were prepared as previously described (Gunzl et al 1995). Gel purified RNA (5000 cpm) was incubated for 30 min on ice in the presence of (10 mM HEPES at pH 7.7, 5 mM MgCl 2 , 1 mM EDTA, 100 mM NaCl) and 30% cell extract or 100 ng recombinant PTB1 protein.…”

Section: Construction Of the Minigene Carrying Aatp11 Trans-splicing mentioning

confidence: 99%

Multiple roles for polypyrimidine tract binding (PTB) proteins in trypanosome RNA metabolism

Stern¹,

Gupta²,

Salmon‐Divon³

et al. 2009

RNA

107

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Trypanosomatid genomes encode for numerous proteins containing an RNA recognition motif (RRM), but the function of most of these proteins in mRNA metabolism is currently unknown. Here, we report the function of two such proteins that we have named PTB1 and PTB2, which resemble the mammalian polypyrimidine tract binding proteins (PTB). RNAi silencing of these factors indicates that both are essential for life. PTB1 and PTB2 reside mostly in the nucleus, but are found in the cytoplasm, as well. Microarray analysis performed on PTB1 and PTB2 RNAi silenced cells indicates that each of these factors differentially affects the transcriptome, thus regulating a different subset of mRNAs. PTB1 and PTB2 substrates were categorized bioinformatically, based on the presence of PTB binding sites in their 59 and 39 flanking sequences. Both proteins were shown to regulate mRNA stability. Interestingly, PTB proteins are essential for trans-splicing of genes containing C-rich polypyrimidine tracts. PTB1, but not PTB2, also affects cis-splicing. The specificity of binding of PTB1 was established in vivo and in vitro using a model substrate. This study demonstrates for the first time that trans-splicing of only certain substrates requires specific factors such as PTB proteins for their splicing. The trypanosome PTB proteins, like their mammalian homologs, represent multivalent RNA binding proteins that regulate mRNAs from their synthesis to degradation.

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Section: Construction Of the Minigene Carrying Aatp11 Trans-splicing mentioning

confidence: 99%

Multiple roles for polypyrimidine tract binding (PTB) proteins in trypanosome RNA metabolism

Stern¹,

Gupta²,

Salmon‐Divon³

et al. 2009

RNA

107

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“…For assaying RNase III activity in crude extracts, cytoplasmic extracts were prepared as previously described (Gunzl et al 1995), spun at 200,000g for 10 min, and frozen in aliquots or used immediately. The protein concentration of the extracts was determined by absorbance at 280 nm.…”

Section: Rnase III Assaysmentioning

confidence: 99%

An unusual Dicer-like1 protein fuels the RNA interference pathway in Trypanosoma brucei

Shi

Tschudi²,

Ullu³

2006

RNA

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RNA interference (RNAi) is an evolutionarily conserved gene-silencing pathway that is triggered by double-stranded RNA (dsRNA). Central to this pathway are two ribonucleases: Dicer, a multidomain RNase III family enzyme that initiates RNAi by generating small interfering RNAs (siRNAs), and Argonaute or Slicer, an RNase H signature enzyme that affects cleavage of mRNA. Previous studies in the early diverging protozoan Trypanosoma brucei have established a key role for Argonaute 1 in RNAi. However, the identity of Dicer has not been resolved. Here, we report the identification and functional characterization of a T. brucei Dicer-like enzyme (TbDcl1). Using genetic and biochemical approaches, we provide evidence that TbDcl1 is required for the generation of siRNA-size molecules and for RNAi. Whereas Dicer and Dicer-like proteins are endowed with two adjacent RNase III domains at the carboxyl terminus (RNase IIIa and RNase IIIb), the arrangement of these two domains is unusual in TbDcl1. RNase IIIa is close to the amino terminus, and RNase IIIb is located approximately in the center of the molecule. This domain organization is specific to trypanosomatids and further illustrates the variable structures of protozoan Dicer-like proteins as compared to fungal and metazoan Dicer.

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“…Nuclear extracts were prepared and in vitro transcription reactions were carried out for L. tarentolae essentially as described for T. brucei (25,26).…”

Section: -And 3-end Analysesmentioning

confidence: 99%

Transcription Termination and 3′-End Processing of the Spliced Leader RNA in Kinetoplastids

Sturm

Campbell

1999

Molecular and Cellular Biology

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Addition of a 39-nucleotide (nt) spliced leader (SL) by trans splicing is a basic requirement for all trypanosome nuclear mRNAs. The SL RNA in Leishmania tarentolae is a 96-nt precursor transcript synthesized by a polymerase that resembles polymerase II most closely. To analyze SL RNA genesis, we mutated SL RNA intron structures and sequence elements: stem-loops II and III, the Sm-binding site, and the downstream T tract. Using an exon-tagged SL RNA gene, we examined the phenotypes produced by a second-site 10-bp linker scan mutagenic series and directed mutagenesis. Here we report that transcription is terminated by the T tract, which is common to the 3 end of all kinetoplastid SL RNA genes, and that more than six T's are required for efficient termination in vivo. We describe mutants whose SL RNAs end in the T tract or appear to lack efficient termination but can generate wild-type 3 ends. Transcriptionally active nuclear extracts show staggered products in the T tract, directed by eight or more T's. The in vivo and in vitro data suggest that SL RNA transcription termination is staggered in the T tract and is followed by nucleolytic processing to generate the mature 3 end. We show that the Sm-binding site and stem-loop III structures are necessary for correct 3-end formation. Thus, we have defined the transcription termination element for the SL RNA gene. The termination mechanism differs from that of vertebrate small nuclear RNA genes and the SL RNA homologue in Ascaris.The spliced leader (SL) RNA is central to kinetoplastid nuclear gene expression. The SL RNA, or mini-exon-derived RNA, is a primary transcript that is synthesized independently of the pre-mRNA and trans spliced onto all nuclear mRNAs (1), most of which are synthesized as polycistronic precursors. SL RNA transcription represents approximately 10% of total RNA synthesis (10,42). The approximately 100 copies of the SL RNA gene are tandemly repeated in a head-to-tail manner in the chromosomal locus; the transcription of each gene is directed by an upstream promoter (26,30,54,62). It has been demonstrated by nuclear run-on analysis that transcription does not proceed into the SL RNA gene-flanking region (40, 62); therefore, the intergenic region (256 bp of a 363-bp repeat in Leishmania tarentolae, 1.2 kb of a 1.35-kb repeat in Trypanosoma brucei) can be considered a nontranscribed spacer (62). The presence of some termination element near the 3Ј end of the SL RNA sequence is thus indicated experimentally.Although the 39-nucleotide (nt) SL exon is well conserved in two domains, the primary sequence of the SL RNA intron is not conserved among the trypanosomes (73). However the secondary structure of the SL RNA, composed of three stemloops and a single-stranded region containing a putative Smbinding site (Fig. 1A), is consistent (11). This structure has been confirmed by physical-chemical and enzymatic studies (29, 44) and examined by mutagenesis (47). An equivalent structure is also conserved in the nematode SL RNAs (11, 53). The Sm-binding site (11)...

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Accurate Transcription of the Trypanosoma brucei U2 Small Nuclear RNA Gene in a Homologous Extract

Cited by 26 publications

References 18 publications

Multiple roles for polypyrimidine tract binding (PTB) proteins in trypanosome RNA metabolism

Multiple roles for polypyrimidine tract binding (PTB) proteins in trypanosome RNA metabolism

An unusual Dicer-like1 protein fuels the RNA interference pathway in Trypanosoma brucei

Transcription Termination and 3′-End Processing of the Spliced Leader RNA in Kinetoplastids

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