Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

Mario Gustavo Mayer,Maria Fernanda Laranjeira Da Silva,Marcos Gonzaga Dos Santos,Lucile Maria Floeter-Winter

doi:10.1590/s0074-02762012000400013

Mario Gustavo Mayer, Maria Fernanda Laranjeira Da Silva + Show 2 more

Open Access

https://doi.org/10.1590/s0074-02762012000400013

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Abstract

The addition of a capped mini-exon [spliced leader (SL)] through trans-splicing is essential for the maturation of RNA polymerase (pol) II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA) are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS) region. Additionally, we detected the SL-5'ETS molecule using three distinct methods and located the acceptor site between two known 5'ETS rRNA processing sites (A' and A1) in four different trypanosomatids. Moreover, we detected a polyadenylated 5'ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin), we observed SL-5'ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA) led to the accumulation of SL-5'ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.

Highlights

Trypanosomatids are a remarkable group of early branching protozoa that have developed unusual strategies to become efficient eukaryotic parasites (Simpson et al 2006)
We have shown that the T. cruzi and Leishmania (Leishmania) amazonensis rRNA pol I promoters can drive the expression of the chloramphenicol acetyltransferase reporter gene, even when no heterologous trans-splice acceptor site is added to the reporter construct (Tyler-Cross et al 1995, Uliana et al 1996, Floeter-Winter et al 1997)
Pre-rRNA is a substrate for trans-splicing in trypanosomatids - To verify the addition of an spliced leader (SL) by trans-splicing in the 5’external transcribed spacer (ETS) regions of pre-rRNAs of four trypanosomatid species (T. brucei, T. cruzi, L. (L.) amazonensis and C. fasciculata), we developed a semi-nested Reverse transcription (RT)-polymerase chain reaction (PCR) strategy (Fig. 2A, B)

Summary

Introduction

Trypanosomatids are a remarkable group of early branching protozoa that have developed unusual strategies to become efficient eukaryotic parasites (Simpson et al 2006). It is not clear if this processing evolved earlier in the metazoan lineage or appeared repeatedly among diverse metazoan lineages (Nilsen 2001) Another unusual feature of the trypanosomatid gene expression repertoire is the use of the powerful RNA pol I machinery to produce large quantities of T. brucei stage-specific variant surface glycoprotein and procyclin precursor (pre-mRNA) (Rudenko et al 1991, Zomerdijk et al 1991). These pre-mRNAs are processed through trans-splicing (Huang & Van der Ploeg 1991). The functional significance of these findings is unclear, we hypothesise that mini-exon addition and/or polyadenylation could serve as tags to drive the destruction of waste molecules in the nuclear compartment

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