When SUMO met splicing

<p>Spliceosomal proteins have been revealed as SUMO conjugation targets. Moreover, we have reported that many of these are in a SUMO-conjugated form when bound to a pre-mRNA substrate during a splicing reaction. We demonstrated that SUMOylation of Prp3 (PRPF3), a component of the U4/U6 di-snRNP, is required for U4/U6•U5 tri-snRNP formation and/or recruitment to active spliceosomes. Expanding upon our previous results, we have shown that the splicing factor SRSF1 stimulates SUMO conjugation to several spliceosomal proteins. Given the relevance of the splicing process, as well as the complex and dynamic nature of its governing machinery, the spliceosome, the molecular mechanisms that modulate its function represent an attractive topic of research. We posit that SUMO conjugation could represent a way of modulating spliceosome assembly and thus, splicing efficiency. How cycles of SUMOylation/de-SUMOylation of spliceosomal proteins become integrated throughout the highly choreographed spliceosomal cycle awaits further investigation.</p

Silencing U5 snRNP components enhances DENV replication.

<p>(A) Reporter DENV replication in A549 cells transfected with siRNA directed to spliceosomal proteins (CD2BP2, DDX23, SNRNP40, PRPF8, EFTUD2, SNRNP200 or SF3A2). An infectious DENV carrying the luciferase gene was used. Three controls were included: a non-related siRNA (NR, black bar), siRNAs directed to Renilla luciferase (control for inhibition, red bar) and siRNA directed to STAT2 (control of NS5 binder with antiviral activity, green bar). Results are representative of three independent experiments (duplicates, mean ± SD). At the bottom, immunoblots indicate the levels of silenced proteins. (B) DENV replication in A549 cells transfected with a non-related siRNA (NR), or siRNAs directed to CD2BP2, DDX23 or EFTUD2, measured by quantitative real time PCR (mean ± SD) at 10hpi for EFTUD2 and 24hpi for CD2BP2 and DDX23. (C) Induced levels of mRNAs corresponding to RIGI, ISG15 and IL8 in A549 cells infected with NDV, which were previously silenced with siRNAs directed to CD2BP2 or EFTUD2 as indicated for each case. RNA levels were measured by quantitative real time PCR (mean ± SD). On the right, immunoblots indicate the levels of silenced proteins and loading controls.</p

NS5 alone interferes with alternative splicing.

<p>(A) NS5 shows a dose dependent effect on the splicing reporter minigenes CFTR, EDI, and Bclx. Radiolabeled amplification products corresponding to different splicing isoforms derived from the indicated minigenes are shown. (B) Quantitative analysis of the changes induced by NS5 (0.9 μg of plasmid). (C) Expression of NS5 mutants with impaired methyltransferase (Mut-MTase) or polymerase (Mut-RdRp) enzymatic activity also alters alternative splicing of reporter genes.</p

DENV infection modifies the splicing landscape of host cells.

<p>(A) Strategy to detect and quantify alternative splicing isoforms in mock infected or DENV infected cells is depicted on the left. Representative autoradiographs and quantification of the inclusion/exclusion ratio for two alternative exon cassette events are shown (duplicates, mean ± SD). (B) Fold-change of inclusion/exclusion ratios for DENV or mock infected cells. A battery of endogenous alternative exon-cassette events (duplicates, mean ± SD) in A549 and Huh7 cells is shown. (C) DENV-induced changes in the splicing pattern of IKKε and MxA mRNAs. Quantification of alternative splicing events is shown for each case.</p

Decreased splicing efficiency in DENV infected cells.

<p>(A) Schematic representation of splicing analysis. The four splicing events analyzed are shown: Intron retention (IR), Exon skipping (ES), Alternative splice site donor/acceptor (Alt5’SS and Alt3’-SS). Constitutive exon and alternative 5’ and 3’SS regions are shown in grey and intron regions in pink. For intron retention the information of the junctions (E1-I, IE-2 and E1E2) was used to calculate the PIR metric. For ES and AltSS the PSI metric was used (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005841#sec012" target="_blank">Materials and methods</a>). (B) Data of splicing analysis of DENV or mock infected cells. On the upper panels, total amount of bins analyzed and altered splicing events for each time point post infection (24 and 36 hours). The percentage of altered events (ES, ALtSS and IR) is shown in pie charts. On the lower panel, increase or decrease retention of introns for each time point is shown.</p

NS5 interferes with an in vitro splicing reaction and inhibits pre-mRNA maturation.

<p>(A) Time course of standard in vitro reaction showing radiolabeled substrate, intermediates and products of the splicing reaction. (B) Purified recombinant NS5 inhibits mature mRNA accumulation. A native or heat denature preparation of NS5 was added to the splicing reaction as indicated on the top of the gel. On the right, a graph showing dose-dependent inhibition of the splicing reaction by NS5. (C) Quantification of pre-mRNA and mature mRNA as a function of time in the presence of native NS5 or heated NS5 at 1μM.</p

Abundance and localization of spliceosomal factors during DENV infection.

<p>(A) Western blots showing the levels of splicing proteins CD2BP2, DDX23, EFTUD2, SNRNP40 or SF3A2 and STAT2 used as a control, at 48 h post-infection. (B) Nuclear localization of CD2BP2 or DDX23 and NS5 in DENV infected or mock infected cells. (C) Determination of relative abundance of U2, U4, U5, and U6 snRNAs in DENV infected or mock A549 and Huh-7 cells.</p