The Leptomonas collosoma spliced leader RNA can switch between two alternate structural forms

LeCuyer, Karen A.; Crothers, Donald M.

doi:10.1021/bi00071a004

Cited by 78 publications

(66 citation statements)

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“…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).…”

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confidence: 54%

“…Secondary structure of the L. tarentolae SL RNA and linker-scanning mutations in the intron. (A) Secondary structure of L. tarentolae SL RNA, based on the L. collosoma structure (form II) that predominates in vivo (29,44); numbering is relative to the start of transcription. The stem-loop structures are labeled I, II, and III, and the exon-intron junction is indicated by the arrow after nt 39.…”

Section: Methodsmentioning

confidence: 99%

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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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confidence: 54%

Section: Methodsmentioning

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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“…During the trans-splicing reaction in trypanosomes and nematodes, spliced leader (SL) RNAs deliver their 5' end as a 5' exon to an independent transcript. The SL RNAs are assembled into ribonucleoprotein particles and have been proposed to adopt secondary structures that bury the 5' splice site in an intramolecular helix (9,36), resembling the interaction of the Ul snRNP with a 5' splice site. Indeed, replacing the 5' splice site region of an adenovirus pre-mRNA with SL RNA sequences from the nematode Caenorhabditis elegans or the trypanosomatid Leptomonas collosoma (C/Adeno or L/Adeno, respectively) allows efficient splicing in HeLa nuclear extract even after the 5' end of Ul is ablated by RNase H in the presence of a DNA oligonucleotide (8).…”

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confidence: 99%

Uncoupling two functions of the U1 small nuclear ribonucleoprotein particle during in vitro splicing.

Seiwert¹,

Steitz²

1993

Mol. Cell. Biol.

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To probe functions of the Ul small nuclear ribonucleoprotein particle (snRNP) during in vitro splicing, we have used unusual splicing substrates which replace the 5' splice site region of an adenovirus substrate with spliced leader (SL) RNA sequences from Leptomonas coUlosoma or Caenorhabditis elegans. In agreement with previous results (J. P. Bruzik and J. A. Steitz, Cell 62:889-899, 1990), we find that oligonucleotide-targeted RNase H destruction of the 5' end of Ul snRNA inhibits the splicing of a standard adenovirus splicing substrate but not of the SL RNA-containing substrates. However, use of an antisense 2'-0-methyl oligoribonucleotide that disrupts the first stem of Ul snRNA as well as stably sequestering positions of Ul snRNA involved in 5' and 3' splice site recognition inhibits the splicing of both the SL constructs and the standard adenovirus substrate. The 2'-0-methyl oligoribonucleotide is no more effective than RNase H pretreatment in preventing pairing of Ul with the 5' splice site, as assessed by inhibition of psoralen cross-link formation between the SL RNA-containing substrate and Ul. The 2'-O-methyl oligoribonucleotide does not alter the protein composition of the Ul monoparticle or deplete the system of essential splicing factors. Native gel analysis indicates that the 2'-O-methyl oligoribonucleotide inhibits splicing by diminishing the formation of splicing complexes. One interpretation of these results is that removal of the 5' end of Ul inhibits base pairing in a different way than sequestering the same sequence with a complementary oligoribonucleotide. Alternatively, our data may indicate that two elements near the 5' end of Ul RNA normally act during spliceosome assembly; the extreme 5' end base pairs with the 5' splice site, while the sequence or structural integrity of stem I is essential for some additional function. It follows that different introns may differ in their use of the repertoire of Ul snRNP functions.Removal of intervening sequences, or introns, from the coding regions of pre-mRNA is catalyzed by a macromolecular complex referred to as the spliceosome. The spliceosome assembles on pre-mRNA from four small nuclear ribonucleoprotein particles (snRNPs) and more than 50 protein factors (for reviews, see references 21, 22, 51, and 62). While functions of only a few of the protein components are known, in vivo and in vitro studies have assigned roles for the Ul, U2, U4, U5, and U6 snRNPs in spliceosome assembly and splicing. This work has indicated that snRNPs are intimately involved in both splice site recognition and determination of the overall architecture of the spliceosome through a network of base pairing and other interactions. The RNA components of the snRNPs have been extensively investigated in part because the similarity between group II self-splicing and pre-mRNA splicing has suggested that these RNAs may be the catalytic component of the spliceosome (12,22,58).Four of the five snRNAs that participate in the splicing reaction use RNA-RNA base pairing to recognize ...

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“…In the mid 1990s, researchers have shown that SL from Leptomonas collosoma and T. brucei displayed two main structures (LeCuyer and Crothers 1993). Similar work in vivo (permeabilized cells) demonstrated that the SL alternates between these structures with predominance of structure 1 (Harris et al 1995).…”

Section: Introductionmentioning

confidence: 97%

Minimum free energy predicted base pairing in the 39 nt spliced leader and 5’ UTR of calmodulin mRNA from Trypanosoma cruzi: influence of the multiple trans-splicing sites

Samudio¹,

Brandão²

2018

An. Acad. Bras. Ciênc.

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We analyzed the compositional changes and the stable base pairs in the predicted secondary structure of the 5' UTR calmodulin mRNA in T. cruzi. The three copies of calmodulin in T. cruzi genome display variable position of the trans splicing sites and give rise to several mRNA that differs slightly on 5' UTR composition in the epimastigote stage. We show that the pattern of high probability base pairs in the minimum free energy predicted secondary structures of the calmodulin 5' UTR remains unchanged despite the nucleotide composition variation. However, the 39 nt spliced leader (mini-exon, the 5' exon sequence transferred to trypanosome mRNAs by the mechanism of trans splicing) shows a variable pattern of high and low probability base pairing as consequence of the altered composition of the 5' UTR. key words: trans-splicing, untranslated region, calmodulin, epimastigote, RNA secondary structure, minimum free energy.

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The Leptomonas collosoma spliced leader RNA can switch between two alternate structural forms

Cited by 78 publications

References 28 publications

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

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

Uncoupling two functions of the U1 small nuclear ribonucleoprotein particle during in vitro splicing.

Minimum free energy predicted base pairing in the 39 nt spliced leader and 5’ UTR of calmodulin mRNA from Trypanosoma cruzi: influence of the multiple trans-splicing sites

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