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

Abstract

The addition of a capped mini-exon [spliced leader (SL)

A small portion of plastid transcripts is polyadenylated in the flagellate Euglena gracilis

“…18S and 28S) rRNAs have not been reported to possess SL-leaders in Euglena. However, a polyA tail is usually missing in nucleus-encoded rRNAs and SL-RNA-mediated processing of nuclear pre-18S rRNA has been recently described in trypanosomatids which are close relatives to euglenids [33]. Moreover, no pathway for rRNA import to plastids is known in euglenids thus far.…”

Identification of SL addition trans-splicing acceptor sites in the internal transcribed spacer I region of pre-rRNA in Leishmania (Leishmania) amazonensis

“…Finally, analysis of the linkage points between the SL and ITS I sequences and the neighbouring sequences clearly indicated that an SL ME was added to the ITS I sequence via canonical trans-splicing. In addition, because we used reverse primers located in the 5.8S region, we can conclude that this processing event occurs prior to the processing between the ITS I and 5.8s RNA regions, suggesting that the trans-splicing at ITS I employs a pre-rRNA as a substrate, instead of a processed spacer.Recently, we described SL addition trans-splicing at the 5'ETS in four trypanosomatid protozoan using a semi-nested RT-PCR approach (Mayer et al 2012). In L. (L.) amazonensis, RNase protection and primer extension experiments validated the semi-nested PCR approach as a valuable tool for detecting true pre-rRNA trans-splicing acceptor sites.…”

“…Taken together, these results reinforce the value of this technique for exploring alternative acceptor molecules for trans-splicing. (Mayer et al 2012) shows that while polyadenylation and trans-splicing are essential for the generation of mature mRNAs, these processing events are not restricted to a specific class of RNA molecules in trypanosomatids.We previously proposed a possible role for transsplicing in a degradation pathway for the 5'ETS region transcribed by RNA polymerase I, although we could not rule out the less probable explanation that this processing event could be a side reaction resulting from spurious RNA pol II transcription of this region (Mayer et al 2012). However, the detection of trans-splicing using another pre-rRNA spacer region, ITS I, as the substrate points to a role for this processing event in the clearance of spacer regions or at least in converting spacer regions into small fragments that are further processed by RNases.…”

“…Moreover, in Trypanosoma brucei, a small number of RNA polymerase II-transcribed tRNA sec precursor transcripts have been shown to exhibit SL ME sequences attached to their 5' ends (Aeby et al 2010). Finally, we have identified pre-rRNA molecules that are processed by transsplicing in the 5' external transcribed spacer (5'ETS) region in four trypanosomatid species [T. brucei, Trypanosoma cruzi, Leishmania (Leishmania) (Mayer et al 2012). These results show that such processing events are not restricted to pre-mRNAs in these organisms.…”

Regulatory mechanisms of Leishmania Aquaglyceroporin AQP1

“…Thus, we examined the stability of AQP1 mRNA in all six species to determine if any post-transcriptional regulation is active in lowering the mRNA levels in the VL species. In order to determine the turnover rate of AQP1 mRNA, mid log phase promastigotes of all species were treated with sinefungin (to stop pre-mRNA processing) (Mayer et al, 2012), followed by actinomycin D (inhibition of transcription) (Mishra et al, 2003), and then harvested up to 130 min. We used quantitative PCR (qPCR) to determine the decay of AQP1 mRNA and normalized against the 0 minute (time of addition of actinomycin D).…”