PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5´-splice-site selection causing tissue-specific defects.

The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5'-splice site (5'SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5'SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches

Atlas of Schistosoma mansoni long non-coding RNAs and their expression correlation to protein-coding genes

Long non-coding RNAs (lncRNAs) have been widely discovered in several organisms with the help of high-throughput RNA sequencing. LncRNAs are over 200 nt-long transcripts that do not have protein-coding (PC) potential, having been reported in model organisms to act mainly on the overall control of PC gene expression. Little is known about the functionality of lncRNAs in evolutionarily ancient non-model metazoan organisms, like Schistosoma mansoni, the parasite that causes schistosomiasis, one of the most prevalent infectious-parasitic diseases worldwide. In a recent transcriptomics effort, we identified thousands of S. mansoni lncRNAs predicted to be functional along the course of parasite development. Here, we present an online catalog of each of the S. mansoni lncRNAs whose expression is correlated to PC genes along the parasite life-cycle, which can be conveniently browsed and downloaded through a new web resource http://verjolab.usp.br. We also provide access now to navigation on the co-expression networks disclosed in our previous publication, where we correlated mRNAs and lncRNAs transcriptional patterns across five life-cycle stages/forms, pinpointing biological processes where lncRNAs might act upon

The Putative <i>Leishmania</i> Telomerase RNA (<i>Leish</i>TER) Undergoes <i>Trans</i>-Splicing and Contains a Conserved Template Sequence

<div><p>Telomerase RNAs (TERs) are highly divergent between species, varying in size and sequence composition. Here, we identify a candidate for the telomerase RNA component of <i>Leishmania</i> genus, which includes species that cause leishmaniasis, a neglected tropical disease. Merging a thorough computational screening combined with RNA-seq evidence, we mapped a non-coding RNA gene localized in a syntenic locus on chromosome 25 of five <i>Leishmania</i> species that shares partial synteny with both <i>Trypanosoma brucei</i> TER locus and a putative TER candidate-containing locus of <i>Crithidia fasciculata</i>. Using target-driven molecular biology approaches, we detected a ∼2,100 nt transcript (<i>Leish</i>TER) that contains a 5′ spliced leader (SL) cap, a putative 3′ polyA tail and a predicted C/D box snoRNA domain. <i>Leish</i>TER is expressed at similar levels in the logarithmic and stationary growth phases of promastigote forms. A 5′SL capped <i>Leish</i>TER co-immunoprecipitated and co-localized with the telomerase protein component (TERT) in a cell cycle-dependent manner. Prediction of its secondary structure strongly suggests the existence of a <i>bona fide</i> single-stranded template sequence and a conserved C[U/C]GUCA motif-containing helix II, representing the template boundary element. This study paves the way for further investigations on the biogenesis of parasite TERT ribonucleoproteins (RNPs) and its role in parasite telomere biology.</p></div

Kaposi’s sarcoma-associated herpesvirus induces specialised ribosomes to efficiently translate viral lytic mRNAs

Viruses rely on the translational machinery of the host cell to synthesis viral proteins. We show that KSHV manipulates the composition of host cell ribosomes creating a specialised ribosome to specifically translate viral mRNAs

Protein acetylation in the critical biological processes in protozoan parasites

Protein lysine acetylation has emerged as a major regulatory post-translational modification in different organisms, present not only on histone proteins affecting chromatin structure and gene expression but also on nonhistone proteins involved in several cellular processes. The same scenario was observed in protozoan parasites after the description of their acetylomes, indicating that acetylation might regulate crucial biological processes in these parasites. The demonstration that glycolytic enzymes are regulated by acetylation in protozoans shows that this modification might regulate several other processes implicated in parasite survival and adaptation during the life cycle, opening the chance to explore the regulatory acetylation machinery of these parasites as drug targets for new treatment development

Extra-telomere BLASTn hits of the TER template sequence on chromosome 25 from the other <i>Leishmania</i> species: <i>L. infantum</i> (LinJ), <i>L. mexicana</i> (LmxM), <i>L. tarentolae</i> (LtaP) and <i>L. braziliensis</i> (LbrM).

<p>All the depicted loci are syntenic to the one in <i>L. major</i> chromosome 25 shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112061#pone-0112061-t001" target="_blank">Table 1</a>.</p><p>Extra-telomere BLASTn hits of the TER template sequence on chromosome 25 from the other <i>Leishmania</i> species: <i>L. infantum</i> (LinJ), <i>L. mexicana</i> (LmxM), <i>L. tarentolae</i> (LtaP) and <i>L. braziliensis</i> (LbrM).</p

<i>Lm</i>TER co-localizes with LmTERT in late S/G2 phase.

<p>RNA FISH coupled with IIF with anti-LaTERT serum. Cells were analyzed throughout the <i>L. major</i> cell cycle. “Merged 1” combines images from LmTERT and <i>Lm</i>TER. “Merged 2” combines all images. Co-localization foci (white arrows) containing <i>Lm</i>TER and LmTERT occur mainly at late S/G2 phases (A). In cells treated with RNase A (B), no RNA hybridization signal or co-localization was detected, indicating that the results shown in (A) correspond to <i>Lm</i>TER and LmTERT co-localizing at the same foci. DAPI (blue) was used to stain DNA in kinetoplast (K) and nucleus (N). These figures contain representative cells of a series of images captured randomly to avoid bias. Scale bar represents 2 µm.</p

The predicted secondary structure model of <i>Lm</i>TER.

<p>A) Proposed secondary structure (mfold - default parameters, followed by visualization through RNAviz editor <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112061#pone.0112061-DeRijk1" target="_blank">[70]</a>) obtained from the first 139 nucleotides of <i>Lm</i>TER (39 nt SL sequence from the 5′ cap processing plus 100 nt from the beginning of the gene in the genome). This folding prediction in that particular region led us to infer the existence of two crucial structured domains already detected in all other TERs reported hitherto: (<i>i</i>) Helix II, containing a CCGUCA motif (red) at its proximal 3′ end, which is implicated in proper template boundary definition in <i>Tetrahymena thermophila</i>; and (<i>ii</i>) the single-stranded template sequence (green). B) The <i>Lm</i>TER structure in A was turned upside-down and compared to the ∼100 nt surrounding template <i>Tb</i>TER structure (the <i>Tb</i>TER sequence used in this analysis and also the default parameters to run the mfold program were identical to those indicated by Gupta and colleagues, 2013). The dashed green box represents the template sequence in <i>Tb</i>TER. Arrows indicate similar shape of hairpin structures immediately downstream of the template on both TERs.</p

Global multiple alignment of the intercoding region on chromosome 25 where <i>LeishTER</i> is located.

<p>Coordinates are relative to the first base after the STOP codon of the respective CDSs: <i>L. major</i> (<i>LmjF.25.0860</i>), <i>L. infantum</i> (<i>LinJ.25.0890</i>), <i>L. mexicana</i> (<i>LmxM.25.0860</i>), <i>L. tarentolae</i> (<i>LtaP25.0910</i>) and <i>L. braziliensis</i> (<i>LbrM.25.0740</i>). The differently shaded colored regions represent the 12 nt template sequence (gray), 5′-C[C/T]GTCA-3′ motif that is part of the template boundary element (TBE) (pink), snoRNA domains found by the RNAspace webserver <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112061#pone.0112061-Cros1" target="_blank">[39]</a> (magenta, the one at position 2216–2259 in LmjF and aligned to other three species is part of snoU90 (or scaRNA7), which is a C/D box snoRNA) and splice acceptor sites (green) detected by RNA-seq (provided by Myler lab and deposited on tritrypdb.org for <i>L. major</i>). ClustalW <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112061#pone.0112061-Thompson1" target="_blank">[27]</a> and Jalview <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112061#pone.0112061-Waterhouse1" target="_blank">[28]</a> were used to align and visualize this locus, respectively. The complete alignment of the whole intercoding region is provided on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112061#pone.0112061.s003" target="_blank">Figure S3</a>.</p