The U6 snRNA-encoding gene of the monogenetic trypanosomatid Leptomonas collosomat

Goldring, Anat; Michaeli, Shulamit

doi:10.1016/0378-1119(95)00048-b

Cited by 18 publications

(8 citation statements)

References 33 publications

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“…Other oligonucleotides were as follows: 21995, 5Ј-GAGGGAGGAAT-GAGGTGAGC-3Ј, complementary to neo mRNA complementary to positions Ϫ1 to ϩ19 (positions 4996 -5016 of the pX-neo vector and position Ϫ1 of the 5Ј-untranslated region) (36); and 12407, 5Ј-AGCTATATCTCTCGAA-3Ј, antisense to the 3Ј-region from positions 83 to 98 of the U6 snRNA gene (37).…”

Section: Methodsmentioning

confidence: 99%

Expression Studies on Clustered Trypanosomatid Box C/D Small Nucleolar RNAs

Xu¹,

Liu²,

López-Estraño³

et al. 2001

Journal of Biological Chemistry

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We analyzed three chromosomal loci of the trypanosomatid Leptomonas collosoma encoding box C/D small nucleolar RNAs (snoRNAs). All the snoRNAs that were analyzed here carry two sequences complementary to rRNA target sites and obey the ؉5 rule for guide methylation. Studies on transgenic parasites carrying the snoRNA-2 gene in the episomal expression vector (pXneo) indicated that no promoter activity was found immediately adjacent to this gene. Deleting the flanking sequences of snoRNA-2 affected the expression; in the absence of the 3-flanking (but not 5-flanking) sequence, the expression was almost completely abolished. The snoRNA genes are transcribed as polycistronic RNA. All snoRNAs can be folded into a common stem-loop structure, which may play a role in processing the polycistronic transcript. snoRNA B2, a member of a snoRNA cluster, was expressed when cloned into the episomal vector, suggesting that each gene within a cluster is individually processed. Studies with permeable cells indicated that snoRNA gene transcription was relatively sensitive to ␣-amanitin, thus supporting transcription by RNA polymerase II. We propose that snoRNA gene expression, similar to protein-coding genes in this family, is regulated at the processing level.

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Section: Methodsmentioning

confidence: 99%

Expression Studies on Clustered Trypanosomatid Box C/D Small Nucleolar RNAs

Xu¹,

Liu²,

López-Estraño³

et al. 2001

Journal of Biological Chemistry

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“…Comparison of the L. seymouri U6 sequence with the trans-spliceosomal U6 RNA from T. brucei (34,46) revealed that all three base changes were located within the 5'-terminal and central domains: in comparison with the T. brucei sequence, the L. seymouri U6 RNA contains an additional G -C base pair at the top of the 5' stem and an A->U change in the central domain (position 25 of T. brucei U6 RNA). Similarly, U6 from L. collosoma differs from the T. brucei sequence at four positions, all residing in the 5'-terminal and central domains (23).…”

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confidence: 99%

trans-spliceosomal U6 RNAs of Crithidia fasciculata and Leptomonas seymouri: deviation from the conserved ACAGAG sequence and potential base pairing with spliced leader RNA.

Wieland

Bindereif

1994

Mol. Cell. Biol.

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U6 RNA genes from the trypanosomatids Crithidia fasciculata and Leptomonas seymouri have been isolated and sequenced. As in Trypanosoma brucei, the U6 RNA genes in both C.fasciculata and L. seymouri are arranged in close linkage with upstream tRNA genes. The U6 RNA sequences from C. fasciculata and L. seymouri deviate in five and three positions, respectively, from the published T. brucei sequence. Interestingly, both C.fasciculata U6 RNA genes carry a C->T change at the second position of the ACAGAG hexanucleotide sequence, which is important for splicing function and has been considered phylogenetically invariable. A compensatory base change of the C. fasciculata spliced leader RNA at the highly conserved 5' splice site position +5, G->A, suggests that an interaction between the 5' splice site region and U6 RNA recently proposed for the yeast cis-splicing system may also occur in trans splicing.Expression of protein-coding genes in trypanosomes involves mRNA processing of a primary transcript, which is usually derived from a long polycistronic transcription unit (for a review, see references 1 and 11). The mature 5' end of each mRNA is generated through trans splicing and the 3' end is generated through polyadenylation. As far as we know, protein-coding genes in trypanosomes are not interrupted by internal introns. Thus there appears to be no conventional cis splicing. Through trans splicing, the 39-nucleotide miniexon sequence at the 5' end of the approximately 140-nucleotide spliced-leader (SL) RNA is joined with each protein-coding exon. trans splicing proceeds through a two-step mechanism of cleavage-ligation reactions, formally analogous to the wellcharacterized cis splicing.A unique component of the trans-splicing machinery is the SL RNA, which occurs in the form of a ribonucleoprotein (RNP) in the cell (13, 31); the core SL RNP is composed of five common proteins, which are also part of the U2 and U4/U6 small nuclear RNPs (snRNPs) (37, 38). The SL RNP has been proposed to play a dual role, functioning as a trans-splicingspecific, possibly Ul-like snRNP and at the same time carrying the miniexon sequence with the 5' splice site (8). How the two trans-splicing substrates are held together within a putative trans spliceosome is unclear; we also know very little about which interactions juxtapose the two splice sites during trans splicing. In the nematode system, there is evidence that an SL-U6 base-pairing interaction is important for trans splicing (25), but it is not clear whether this interaction is conserved in the trypanosome system. U6 RNA undergoes several conformational transitions during the spliceosome cycle. First, it is present in a singular form (39); second, in the U4/U6 snRNP it is stably base paired with U4 RNA (7); third, after the U4-U6 interaction has been disrupted in the spliceosome, U6 interacts by base pairing with U2, which is considered the active conformation of U6 (30). Many studies of the yeast and mammalian cis-splicing systems have provided evidence that the U6 RNA is one of t...

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“…In nematodes, U6 seems to have a unique function since it interacts with the SL RNA in the Sm site and 3 nt downstream from it, serving as a bridge between the SL RNA and the pre-mRNA (31). Although an analogous interaction can be envisioned in different trypanosomatid species (26), no functional proof of such interaction was obtained, since mutations in 3 nt downstream from the Sm site that affected trans splicing in the nematodes had no severe trans-splicing defect in L. collosoma (53). These data suggest that trypanosomes and nematodes differ in the mechanism by which the SL RNA is brought into the spliceosome.…”

Section: Sl-snrna Interactionsmentioning

confidence: 97%

“…Because of space limitation, we chose to discuss snRNAs that have unique features in trypanosomes. Detailed information on U6 and U4 from different trypanosomatid species is not covered in this review but can be found in (8,26,44,105).…”

Section: Snrnas Involved In Trans and Cis Splicingmentioning

confidence: 99%

trans and cis Splicing in Trypanosomatids: Mechanism, Factors, and Regulation

et al. 2003

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mRNA maturation in trypanosomes differs from the process in most eukaryotes mainly because protein-coding genes are transcribed into polycistronic RNAs in this organism (78). Studies from the ongoing genome project suggest that the entire chromosome may be transcribed as large transcripts, but thus far there is no evidence to support the existence of conventional polymerase II promoters (62). The process of trans splicing was discovered 20 years ago when it was found that the different variant surface glycoprotein mRNAs in Trypanosoma brucei carry a common 39-nucleotide (nt) sequence, namely, the spliced leader (SL) sequence (9). It was later established that all trypanosome mRNAs undergo trans splicing (2). The source of the SL sequence was found to be a small capped RNA, the SL RNA (14, 58). Thus, the SL addition serves two purposes: it functions together with polyadenylation in dissecting the polycistronic transcripts, and it provides the cap to mRNAs (2). trans splicing proceeds through a two-step transesterification reaction, analogous to cis splicing but forming a Y structure instead of a lariat intermediate (illustrated in Fig. 1A) (61,88). Although first discovered in trypanosomes, the process was later found in nematodes (41), euglenoids (91), trematodes (73), and recently in chordates (97). Surprisingly, after almost a decade of searching for cis splicing, a single gene carrying a cis-spliced intron was discovered, suggesting that these two splicing processes coexist in trypanosomes, as in all other organisms capable of trans splicing (51).In the last decade, studies have focused on elucidating the mechanism and machinery of pre-mRNA processing in these organisms. The major findings included (i) the identification of the first cis-spliced intron and U1 snRNA that may function exclusively in this process, (ii) the finding and unraveling the function of U5 and SLA1, and (iii) the existence of coupling between trans splicing and polyadenylation. In this review we summarize studies performed mainly in vivo to elucidate structure-function aspects of the SL RNA and the snRNAs with which it interacts. The unique modifications on the SL RNA, including capping and pseudouridylation and their role in SL RNA function and biogenesis, are described. The regulation of splicing and its linkage to polyadenylation is discussed, and data are provided for the existence of splicing factors whose function is well characterized in other eukaryotes. STRUCTURE-FUNCTION ANALYSIS OF THE SL RNAIn the absence of an in vitro system for trans splicing, most of the structure-function studies were performed in vivo in Leptomonas seymouri, Leptomonas collosoma, and Leishmania tarentolae by using tagged SL RNAs (49,53,85,86,108). For some unknown reason, this approach does not work for T. brucei (Elisabetta Ullu, unpublished data). The SL RNA secondary structure of all organisms carrying out trans splicing is similar; it is composed of three stem-loops. In trypanosomatids, The SL sequence is 39 to 41 nt, followed by an intron of varia...

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The U6 snRNA-encoding gene of the monogenetic trypanosomatid Leptomonas collosomat

Cited by 18 publications

References 33 publications

Expression Studies on Clustered Trypanosomatid Box C/D Small Nucleolar RNAs

Expression Studies on Clustered Trypanosomatid Box C/D Small Nucleolar RNAs

trans-spliceosomal U6 RNAs of Crithidia fasciculata and Leptomonas seymouri: deviation from the conserved ACAGAG sequence and potential base pairing with spliced leader RNA.

trans and cis Splicing in Trypanosomatids: Mechanism, Factors, and Regulation

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