The vault RNA of Trypanosoma brucei plays a role in the production of trans-spliced mRNA

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.

Keywords: RNA; RNA metabolism; RNA processing; RNA splicing; RNA-binding protein; Ro protein; Trypanosoma brucei; spliced leader (SL); telomerase-associated protein 1 (TEP1); vault RNA.

Figure 1.

TBsRNA-10 is the T. brucei vtRNA. A, Northern blot analysis of total T. brucei RNA probed with ³²P-labeled antisense TBsRNA-10 oligodeoxyribonucleotide probe. Marker is a ³²P-labeled pBR322 DNA MspI digest. B, predicted secondary structure of the T. brucei vtRNA. Shown as double-stranded are only the terminal helix and very stable hairpins emanating from the large internal loop. Evolutionarily conserved residues (see Fig. 3) are highlighted. C, diagram outlining the domain architecture of human and trypanosome TEP1. D, Northern blot analysis of the indicated RNAs after immunoprecipitation with anti-TEP1 sera from two rabbits (Rbt1 and Rbt2). The fractions of the input and precipitated RNA loaded are indicated with percentages. E, three-dimensional structure models for the TROVE domains of human (top) and T. brucei (bottom) TEP1 obtained with SWISS-MODEL (93) based on the crystal structure of X. laevis Ro60 protein (30). The vWFA domains are included in the models.

Figure 2.

TEP1 RNAi silencing affects vtRNA levels. A, reduction of vtRNA levels upon silencing TEP1. Cells carrying the RNAi construct for TEP1 were induced for the indicated number of days, and total RNA was isolated and subjected to Northern blot analysis with the indicated probes. B, graphical representation of the results from four independent TEP1 RNAi experiments showing the levels of vtRNA relative to 7SL, SL, U4, and SLA1 RNA. The individual data points (symbols) for all experiments are presented, and the line connects the average values ±S.D.

Figure 3.

Sequence alignment of the newly identified vtRNAs from trypanosomatids with selected vtRNAs from other eukaryotes. Shown are the sequences of the vtRNA genes. Species with more than one vtRNA are represented by a single example. The examples from H. sapiens to S. kowalevskii are from Ref. , and the remaining genes are newly identified. The positions of the internal box A and box B transcription elements are indicated, and nucleotides that are at least 60% conserved are highlighted. Arrows indicate the two strands of the bulged terminal helix.

Figure 4.

T. brucei vtRNA is transcribed by RNA polymerase III. The plots show the number of ChIP-seq reads aligning to specific genomic locations for either the input (blue) or the anti-pol III-immunoprecipitated (red) material. A, shown are the chromosome regions containing the vtRNA gene; B, U2 snRNA gene; C, U1 snRNA, U3 snoRNA, 7SL RNA, ant tRNAs genes; D, U4 snRNA and a tRNA gene; E, U6 snRNA and tRNAs genes; F, U5 snRNAs and tRNAs genes; G, example with tRNA genes only. The location of the small RNA genes is indicated with red arrowheads according to their orientation. The locations of protein-coding sequences are indicated with gray arrows. Gene accession numbers are shown only for the two genes neighboring the pol III transcription units.

Figure 5.

Two regions in T. brucei vtRNA are accessible for base-pairing interactions. A, diagram depicting the antisense oligodeoxyribonucleotides used in the RNase H cleavage assay. Shown is the antisense probe used for Northern blotting. B, Northern blot analysis detecting vtRNA in the samples treated with RNase H and the indicated oligos. Controls without oligo (+RNase H) and without both oligo and RNase H (−RNase H) are loaded at the beginning. Marker is a ³²P-labeled pBR322 DNA MspI digest.

Figure 6.

T. brucei vtRNA is highly enriched in a nuclear compartment distinct from the nucleolus. High-resolution fluorescence in situ hybridization coupled with immunofluorescence was performed for the indicated small RNAs (green) and NHP2 (red), top panels. DNA was stained with DAPI (blue). Bottom panels, simultaneous detection by immunofluorescence of NHP2 (red) and MTAP (blue) with in situ hybridization for the indicated RNAs (green). Insets show three-dimensional representation of the detected RNA and proteins.

Figure 7.

vtRNA in T. brucei is required for the production of trans-spliced mRNA. A, diagram of the mechanism of coupled trans-splicing and polyadenylation in T. brucei. Note that the timing of splicing and upstream mRNA polyadenylation is currently unknown. Efficiency of processing events upstream or downstream of any mRNA could result in species that are either not spliced or not polyadenylated. B, Northern blot analysis of the indicated RNAs in permeabilized BF cells treated with the vtRNA antisense oligodeoxynucleotide VT-A or VT-B or their mutant versions, MUT-A or MUT-B, as controls. C, analysis of newly synthesized, ³²P-labeled RNA in permeabilized BF cells treated with MUT-A or VT-A oligo. Input is the total cellular RNA; SLex samples represent the RNA enriched on streptavidin beads coupled to biotinylated antisense oligo against the SL exon sequence; poly(A) is the RNA enriched on beads with biotinylated (dT)₃₉ oligonucleotide; D, as in C, but for cells treated with the MUT-B or VT-B oligo. Marker is a ³²P-labeled pBR322 DNA MspI digest. Indicated are the positions of the SL RNA and SL RNA fragments. As the conditions for the assay were optimized for pol II transcription, the majority of the high-molecular-weight signal represents pre-mRNA and mRNA.

Figure 8.

Effect of reduction of vtRNA levels on the newly synthesized SL RNA after longer RNA synthesis time in permeabilized cells. A, analysis of newly-synthesized, ³²P-labeled RNA in permeabilized BF cells treated with the MUT-A or VT-A antisense oligos after a 30-min incubation with ribonucleoside triphosphate mixture containing [α-³²P]UTP. SLex, RNA enriched on antisense SL oligonucleotide beads; poly(A), RNA enriched on (dT)₃₉ beads. The positions of pre-mRNA/mRNA, SL RNA, the 3′-end–shortened SL 130 RNA, and the SL RNA fragments are indicated. B, Northern blot analysis of the indicated RNAs in permeabilized cells treated with the MUT-A or VT-A oligos, after incubation for 30 min with only nonradioactive ribonucleoside triphosphates. C, newly synthesized, ³²P-labeled RNA in permeabilized cells treated with the MUT-A or VT-A antisense oligos after a 5-min incubation with ribonucleoside triphosphate mixture containing [α-³²P]UTP. Input, total RNA in the cells; SLin, RNA enriched on beads coupled to antisense oligonucleotide against the SL RNA intron.