Abstract
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 (pX-neo) 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 alpha-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.
Highlights
The nucleolus of eukaryotic cells contains numerous small nucleolar RNAs1 that play a regulatory role in rRNA biogenesis [1,2,3,4,5]
Tel.: 972-3-531-8068; Fax: 972-3-535-1824; E-mail: michaes@mail.biu.ac.il. 1 The abbreviations used are: snoRNAs, small nucleolar RNAs; pol, polymerase; snRNA, small nuclear RNA; bp, base pair(s); kb, kilobase; PCR, polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; SL, spliced leader. Both yeast and mammals indicate that the fifth nucleotide (ϩ5) upstream from box D or DЈ within the domains of interaction between the guide RNA and the rRNA target site is methylated; this is known as the ϩ5 rule [7, 10, 13]
Our analyses indicate that there is no irregularity in the guide methylation rule of the trypanosomatid snoRNAs and that the L. collosoma box C/D snoRNAs obey the ϩ5 rule
Summary
C/D snoRNAs were analyzed from T. brucei [35] by immunoprecipitation using antibodies against the cloned T. brucei fibrillarin protein. Mapping of the methylation sites potentially guided by eight snoRNAs suggested that the T. brucei snoRNAs obey the ϩ5 rule. To further elucidate the trypanosomatid snoRNA structurefunction relationship and the mode of transcription and processing, we studied four box C/D snoRNAs encoded by three chromosomal loci. Our analyses indicate that there is no irregularity in the guide methylation rule of the trypanosomatid snoRNAs and that the L. collosoma box C/D snoRNAs obey the ϩ5 rule. Proper expression of a tagged snoRNA-2 gene cloned into the episomal vector pX-neo requires at least 20-nucleotide flanking sequences. Expression, at a lower level, was detected even in the absence of an upstream sequence, suggesting the lack of a conventional promoter adjacent to the gene. Transcriptional studies performed in permeable cells suggest that snoRNA gene transcription is relatively sensitive to ␣-amanitin, similar to pol II-transcribed genes.
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