In vivo transcriptional analysis of the spliced leader RNA gene in the trypanosomatid Leptomonas seymouri

Crenshaw-Williams, Karla; Bellofatto, Vivian

doi:10.1007/s004360050618

Cited by 3 publications

(4 citation statements)

References 23 publications

(32 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The ambiguous results of drug studies have made it extremely difficult to determine the identity of the RNA polymerase responsible for transcription of the SL RNA gene. In permeabilized trypanosomes, mRNA genes (notably α-tubulin) were downregulated by 5-10 µg/ml α-amanitin and rRNA genes required substantially more (500-1000 µg/ml) (26,47). RNA pol III-dependent small RNAs, including many of the U snRNAs, tRNA and 5S RNA, had the expected intermediate α-amanitin sensitivity; 100-200 µg/ml blocked transcription.…”

Section: Discussionmentioning

confidence: 97%

“…Tagetitoxin, a potent inhibitor of RNA pol III activity, had no effect on SL RNA transcription (4,25). However, α-amanitin, a low-dose inhibitor of RNA pol II, inhibited SL RNA transcription at doses higher than needed for inhibition of α-tubulin, a prototypic RNA pol II gene (3,4,26,27). The SL RNA transcript is generally not polyadenylated (28,29) and termination of SL RNA transcription requires an intact 3′ poly(T) tract, both hallmarks of RNA pol III-dependent genes (30).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Trypanosome spliced leader RNA genes contain the first identified RNA polymerase II gene promoter in these organisms

Gilinger

2001

Nucleic Acids Research

110

View full text Add to dashboard Cite

Typical general transcription factors, such as TATA binding protein and TFII B, have not yet been identified in any member of the Trypanosomatidae family of parasitic protozoa. Interestingly, mRNA coding genes do not appear to have discrete transcriptional start sites, although in most cases they require an RNA polymerase that has the biochemical properties of eukaryotic RNA polymerase II. A discrete transcription initiation site may not be necessary for mRNA synthesis since the sequences upstream of each transcribed coding region are trimmed from the nascent transcript when a short m(7)G-capped RNA is added during mRNA maturation. This short 39 nt m(7)G-capped RNA, the spliced leader (SL) sequence, is expressed as an approximately 100 nt long RNA from a set of reiterated, though independently transcribed, genes in the trypanosome genome. Punctuation of the 5' end of mRNAs by a m(7)G cap-containing spliced leader is a developing theme in the lower eukaryotic world; organisms as diverse as EUGLENA: and nematode worms, including Caenorhabditis elegans, utilize SL RNA in their mRNA maturation programs. Towards understanding the coordination of SL RNA and mRNA expression in trypanosomes, we have begun by characterizing SL RNA gene expression in the model trypanosome Leptomonas seymouri. Using a homologous in vitro transcription system, we demonstrate in this study that the SL RNA is transcribed by RNA polymerase II. During SL RNA transcription, accurate initiation is determined by an initiator element with a loose consensus of CYAC/AYR(+1). This element, as well as two additional basal promoter elements, is divergent in sequence from the basal transcription elements seen in other eukaryotic gene promoters. We show here that the in vitro transcription extract contains a binding activity that is specific for the initiator element and thus may participate in recruiting RNA polymerase II to the SL RNA gene promoter.

show abstract

Section: Discussionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

Trypanosome spliced leader RNA genes contain the first identified RNA polymerase II gene promoter in these organisms

Gilinger

2001

Nucleic Acids Research

110

View full text Add to dashboard Cite

show abstract

“…Transcriptional analysis of SL RNA genes in three related trypanosomatids have shown that alterations within the SL RNA sequence had minor effects on both transcription efficiency and start site selection, although these effects were not studied in detail (5,17,27). As an important component of our Inr t studies, we determined directly if the SL RNA sequence must follow the Inr t immediately.…”

Section: In Vitro Transcription Analyses Identify Promoter Elementmentioning

confidence: 99%

“…This assignment is based on transcription elongation inhibition studies using ␣-amanitin, in which the inhibition pattern of the SL RNA most closely resembles, but does not overlap, the mRNA inhibition profile. RNA pol II-dependent mRNA gene promoters elude definition in trypanosomatids (14,17,18,27). The sequence-specific binding of PBP-1 and PBP-2 to two core SL RNA gene promoter elements argues that a subset of RNA pol II genes functions by recruiting specific DNA-binding proteins upstream from a discrete start site.…”

Section: In Vitro Transcription Analyses Identify Promoter Elementmentioning

confidence: 99%

Transcription Initiation at the TATA-less Spliced Leader RNA Gene Promoter Requires at Least Two DNA-binding Proteins and a Tripartite Architecture That Includes an Initiator Element

Luo

Gilinger

Mukherjee

et al. 1999

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Eukaryotic transcriptional regulatory signals, defined as core and activator promoter elements, have yet to be identified in the earliest diverging group of eukaryotes, the primitive protozoans, which include the Trypanosomatidae family of parasites. The divergence within this family is highlighted by the apparent absence of the "universal" transcription factor TATA-binding protein. To understand gene expression in these protists, we have investigated spliced leader RNA gene transcription. The RNA product of this gene provides an m 7 G cap and a 39-nucleotide leader sequence to all cellular mRNAs via a trans-splicing reaction. Regulation of spliced leader RNA synthesis is controlled by a tripartite promoter located exclusively upstream from the transcription start site. Proteins PBP-1 and PBP-2 bind to two of the three promoter elements in the trypanosomatid Leptomonas seymouri. They represent the first trypanosome transcription factors with typical doublestranded DNA binding site recognition. These proteins ensure efficient transcription. However, accurate initiation is determined an initiator element with a a loose consensus of CYAC/AYR (؉1), which differs from that found in metazoan initiator elements as well as from that identified in one of the earliest diverging protozoans, Trichomonas vaginalis. Trypanosomes may utilize initiator element-protein interactions, and not TATA sequence-TATA-binding protein interactions, to direct proper transcription initiation by RNA polymerase II.Molecular studies of trypanosomatids, a ubiquitous and diverse family of protozoan pathogens, have revealed strikingly unusual mechanisms of mRNA synthesis. One central device is that two independent transcription events direct each mRNA produced in the trypanosome nucleus (for review, see Ref. 1). The protein-coding portion is transcribed as a single primary mRNA, often containing several open reading frames flanked by 5Ј-and 3Ј-untranslated regions. The capped 5Ј-end portion is transcribed as a short spliced leader (SL) 1 RNA. The two parts are fused in a trans-splicing reaction that yields a functional mRNA. During fusion, the 39 nt present on the 5Ј-end of the SL RNA (and referred to as the SL) are transferred to a region upstream from the coding region on the primary mRNA (2). Addition of the SL provides each mRNA with an m 7 G cap as well as four extensively methylated nucleotides, at positions 1-4 within the 39-nt SL RNA (3).The SL RNA is transcribed from a highly reiterated set of genes. In contrast to the long primary transcripts that form the bulk of the mature mRNA, each SL RNA has a discrete transcriptional start site. ␣-Amanitin studies show that it is very probable, though not proven, that the SL RNA gene is transcribed by RNA polymerase (pol) II. The primary SL RNA transcript and the transcript present in the trans-splicing spliceosome possess identical 5Ј-and 3Ј-ends, indicating that both transcription initiation and termination regulate the accumulation of SL RNA. SL RNA expression has been monitored using independe...

show abstract

In vitro transcription of mutated Leishmania tarentolae spliced leader RNA genes approximates in vivo patterns

Roberts

Sturm

et al. 2000

Molecular and Biochemical Parasitology

View full text Add to dashboard Cite

In vivo transcriptional analysis of the spliced leader RNA gene in the trypanosomatid Leptomonas seymouri

Cited by 3 publications

References 23 publications

Trypanosome spliced leader RNA genes contain the first identified RNA polymerase II gene promoter in these organisms

Trypanosome spliced leader RNA genes contain the first identified RNA polymerase II gene promoter in these organisms

Transcription Initiation at the TATA-less Spliced Leader RNA Gene Promoter Requires at Least Two DNA-binding Proteins and a Tripartite Architecture That Includes an Initiator Element

In vitro transcription of mutated Leishmania tarentolae spliced leader RNA genes approximates in vivo patterns

Contact Info

Product

Resources

About