A new role for vault RNA–TEP1 complexes in mRNA production in trypanosomes

Abstract

Vault RNAs, found in vault ribonucleoprotein complexes, are known to be one of many types of small noncoding RNAs (ncRNAs), but their specific function is not known. A new study identifies a small ncRNA from Trypanosome brucei as a vault RNA (vtRNA) based on sequence analysis and its association with the canonical vault component TEP1. Down-regulation of T. brucei vtRNA impairs mRNA splicing in a permeabilized cell system, suggesting new roles for these enigmatic biomolecules.

Introduction

Most eukaryotes contain a poorly understood organelle called the vault; each copy of this large (13-MDa) cytoplasmic particle is composed of 78 copies of the major vault protein (MVP),² fewer copies of vault poly(ADP-ribose) polymerase, the telomerase-associated protein (TEP1), and vault RNA (vtRNA) (1). TEP1 is responsible for vault RNA association with the particle (2). Because not all cells contain vault particles, and knockouts of the MVP have little biological effect, assigning specific functions to the vault has been difficult. vtRNA function, like that of the vault particle, has been elusive. Over the years, the human vtRNAs (predominantly human vtRNA1-1) have been associated with numerous cellular events, including being processed into small regulatory RNAs; up-regulation and anti-viral activity in response to some specific viruses; regulation of apoptosis; exosome-associated intercellular communication; and multidrug resistance. More recently, novel additional vtRNA-protein complexes (vtRNPs) have been uncovered that are not known to interact with the vault particles. Specifically, the vtRNA1-1 was shown to act as riboregulator of autophagy via its interactions with a particular receptor (3). Moreover, cytosine methylation of vtRNA nucleotide 69 appears to regulate its interaction with a splicing factor, altering epidermal cell differentiation (4). However, a complete understanding of these functional associations and their conservation throughout evolution awaits discovery. A new study of the early eukaryote Trypanosome brucei sheds light on another system by confirming the trypanosomal homologues of the vtRNA and TEP1 pair and identifying a possible role for the vtRNP in RNA processing. These results bridge a gap between the function of this vtRNP, outside of the vault particle, and its known association with the vault particle.

vtRNAs are small noncoding RNAs transcribed by RNA polymerase III, and they vary in length from 80 to 140 bases depending on the species. Only a small fraction of total cellular vtRNA sediments as part of the vault particle (5), where it is bound into the particle in a subcomplex with TEP1. In TEP1 knockout mice, the vtRNA does not associate with vault particles, and the levels of vtRNA are reduced (2). The fact that some vtRNA remains, indicates that vtRNA is only partially stabilized by interacting with TEP1 and implies that the remaining nonparticulate vtRNA is stabilized by other unidentified non-vault-associated proteins or unidentified RNAs via base-pairing. vtRNAs can be folded into a secondary structure that is similar to a second class of RNAs called Y RNAs, which, in complex with the Ro protein, are involved in RNA processing. Both Ro and TEP1 proteins contain the unique TROVE domain responsible for RNA binding (6).

Previously, a high-throughput screen of small RNAs from the unicellular organism T. brucei identified an abundant RNA (TBxRN-10) that localizes to a nonnucleolar region of the nucleus enriched in the bloodstream form of the parasitic organism (5). In this issue, Kolev and colleagues now explore this RNA in more detail, first noticing that the predicted TBxRN-10 secondary structure resembles that of Y RNAs and vtRNAs (7). A search of the T. brucei genome for Ro and TEP1 protein homologues identified a candidate TEP1 protein (Tb927.783), but no Ro protein homolog, suggesting that the sequence is a vtRNA. Immunoprecipitation using antibodies raised against C-terminal peptides of the putative T. brucei TEP1 demonstrated that the vtRNA was quantitatively bound to the protein. This is the first time that the RNP complex of TEP1 and vtRNA has been isolated separately from the vault complex. As in the TEP1 knockout mice, knockdown of T. brucei TEP1 reduced vtRNA levels. ChIP-Seq analysis revealed that the trypanosome vtRNA genes are transcribed by RNA Pol III and that the promoter elements are located upstream of the gene, resembling the architecture of other genes transcribed by Pol III in trypanosomes. Bioinformatics analysis pointed to the presence of candidate vtRNA genes in all sequenced trypanosomes, with the presence of one vtRNA confirmed in Leishmania braziliensis, suggesting the generality of these findings. Furthermore, the authors noted that vtRNAs across species share a highly conserved sequence. A probe of vtRNA secondary structure using an RNase H–directed oligodeoxynucleotide cleavage assay revealed that these sequences in the T. brucei TEP1-associated vtRNA are in an open conformation and thus available for base-pairing with other cellular RNAs.

Next, Kolev et al. considered the intracellular location of this new sequence. Mammalian vtRNA is primarily cytoplasmic, but a portion is found in the nucleus. In contrast, T. brucei vtRNA localizes exclusively to a nuclear subdomain that is enriched in the RNAs and proteins necessary for trans-splicing, a process used by lower eukaryotes in place of the intron-based cis-splicing to modify mRNA sequences. The authors hypothesized that vtRNPs may facilitate the biogenesis of the incoming RNA element known as the splice leader (SL) and thus mediate trans-splicing. Using an optimized, cell-permeabilized system of T. brucei, the authors showed that oligonucleotide-directed cleavage of vtRNA decreased the production of the SL exon-enriched long RNA products, leading the authors to conclude that the vtRNA/TEP1 RNP is indeed involved in the production of SL trans-spliced mRNA in this organism. This could be an indirect effect, as they were unable to detect direct interactions between TEP1 and/or vtRNA and the trans-splicing machinery.

The new results from Kolev et al. provide several immediate opportunities to learn more about these interesting systems. First, the isolation of the vtRNP enables new structural questions to be asked. For example, it is not clear how the vtRNP complex could interact with vault particles (i.e. does it cycle in and out?). Given that the MVP is not necessary for cell survival, could the vault particle regulate other vtRNA-RNPs by sequestering vtRNA away from them? Do trypanosomes contain vault particles? Its genome contains putative homologues of the defining vault protein MVP, so this is likely to be the case. Second, the connection to trans-splicing opens up new directions in exploring RNA processing. Does the vtRNP function in trypanosomes translate to a similar role in more complex organisms? If so, this humble organism may open a new door to understanding vault particle function.