Abstract
The vault ribonucleoprotein (RNP), comprising vault RNA (vtRNA) and telomerase-associated protein 1 (TEP1), is found in many eukaryotes. However, previous studies of vtRNAs, for example in mammalian cells, have failed to reach a definitive conclusion about their function. vtRNAs are related to Y RNAs, which are complexed with Ro protein and influence Ro's function in noncoding RNA (ncRNA) quality control and processing. In Trypanosoma brucei, the small noncoding TBsRNA-10 was first described in a survey of the ncRNA repertoire in this organism. Here, we report that TBsRNA-10 in T. brucei is a vtRNA, based on its association with TEP1 and sequence similarity to those of other known and predicted vtRNAs. We observed that like vtRNAs in other species, TBsRNA-10 is transcribed by RNA polymerase III, which in trypanosomes also generates the spliceosomal U-rich small nuclear RNAs. In T. brucei, spliced leader (SL)-mediated trans-splicing of pre-mRNAs is an obligatory step in gene expression, and we found here that T. brucei's vtRNA is highly enriched in a non-nucleolar locus in the cell nucleus implicated in SL RNP biogenesis. Using a newly developed permeabilized cell system for the bloodstream form of T. brucei, we show that down-regulated vtRNA levels impair trans-spliced mRNA production, consistent with a role of vtRNA in trypanosome mRNA metabolism. Our results suggest a common theme for the functions of vtRNAs and Y RNAs. We conclude that by complexing with their protein-binding partners TEP1 and Ro, respectively, these two RNA species modulate the metabolism of various RNA classes.
Highlights
The vault ribonucleoprotein (RNP), comprising vault RNA and telomerase-associated protein 1 (TEP1), is found in many eukaryotes
Vault RNAs3 are small noncoding RNAs first identified as components of the large barrel-shaped cytoplasmic vault structures in eukaryotes [1]. vault RNA (vtRNA) are transcribed by RNA polymerase III [2] and vary in length from 80 to 140 nt [3]
We found that the vtRNA in T. brucei affects the production of trans-spliced mRNAs, and our data suggest a similarity between the function of the vtRNA in trypanosomatids and the functions of Y RNAs in other species in controlling the metabolism of RNA molecules
Summary
An abundant noncoding RNA, named TBsRNA-10 (Fig. 1, A and B), was discovered in a high-throughput sequencing survey of T. brucei small RNAs [45]. We observed very efficient enrichment of fragments corresponding to the SL exon sequence among the RNAs selected by the anti-SL oligo, even though they were barely detectable in the total input RNA (Fig. 7, C and D) The abundance of these SL exon fragments was different between cells treated with anti-vtRNA oligos VT-A and VT-B and was less prominent in the anti-vtRNA samples by 40% for both oligos (Fig. 7, C and D). Very similar results were obtained with newly-synthesized RNA for 30 min (Fig. 8A), a 75% decrease of SL-containing long RNAs in the anti-vtRNA cells, a 15% decrease in polyadenylated species, which again appeared shorter compared with mutant oligo-treated cell RNAs, and a 45% decrease in the abundance of the SL exon fragments (Fig. 8A). We detected a shift in the size of high-molecular-weight newly-synthesized RNA containing the branched SL intron toward shorter species (Fig. 8C)
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