G-quadruplexes are widespread four-stranded structures that are adopted by G-rich regions of both DNA and RNA and are involved in essential biological processes such as mRNA translation. They are formed by the stacking of two or more G-quartets that are linked together by three loops. Although the maximal loop length is usually fixed to 7 nt in most G-quadruplexpredicting software, it has already been demonstrated that artificial DNA G-quadruplexes containing two distal loops that are limited to 1 nt each and a central loop up to 30 nt long are likely to form in vitro. This report demonstrates that such structures possessing a long central loop are actually found in the 5 ′ -UTRs of human mRNAs. Firstly, 1453 potential G-quadruplex-forming sequences (PG4s) were identified through a bioinformatic survey that searched for sequences respecting the requirement for two 1-nt long distal loops and a long central loop of 2-90 nt in length. Secondly, in vitro in-line probing experiments confirmed and characterized the folding of eight candidates possessing central loops of 10-70 nt long. Finally, the biological effect of several G-quadruplexes with a long central loop on mRNA expression was studied in cellulo using a luciferase gene reporter assay. Clearly, the actual definition of G-quadruplex-forming sequences is too conservative and must be expanded to include the long central loop. This greatly expands the number of expected PG4s in the transcriptome. Consideration of these new candidates might aid in elucidating the potentially important biological implications of the G-quadruplex structure.

ABX464 is a first-in-class, clinical-stage, small molecule for oral administration that has shown strong anti-inflammatory effects in the DSS-model for inflammatory bowel disease (IBD) and also prevents replication of the HIV virus. ABX464 which binds to cap binding complex (CBC) has demonstrated safety and efficacy in a phase 2a proof-of-concept clinical trial in patients with Ulcerative colitis. Previously, with limited technologies, it was not possible to quantify the effect of ABX464 on viral and cellular RNA biogenesis. Here, using RNA CaptureSeq and deep sequencing, we report that ABX464 enhances the splicing of HIV RNA in infected PBMCs from six healthy individuals and also the expression and splicing of a single long noncoding RNA to generate the anti-inflammatory miR-124 both ex vivo and in HIV patients. While ABX464 has no effect on pre-mRNA splicing of cellular genes, depletion of CBC complex by RNAi leads to accumulation of intron retention transcripts. These results imply that ABX464 did not inhibit the function of CBC in splicing but rather strengthens it under pathological condition like inflammation and HIV infection. The specific dual ability of ABX464 to generate both anti-inflammatory miR-124 and spliced viral RNA may have applicability for the treatment of both inflammatory diseases and HIV infection.

We investigated the molecular mechanisms for in-frame skipping of DMD exon 39 caused by the nonsense c.5480T>A mutation in a patient with Becker muscular dystrophy. RNase-assisted pull down assay coupled with mass spectrometry revealed that the mutant RNA probe specifically recruits hnRNPA1, hnRNPA2/B1 and DAZAP1. Functional studies in a human myoblast cell line transfected with DMD minigenes confirmed the splicing inhibitory activity of hnRNPA1 and hnRNPA2/B1, and showed that DAZAP1, also known to activate splicing, acts negatively in the context of the mutated exon 39. Furthermore, we uncovered that recognition of endogenous DMD exon 39 in muscle cells is promoted by FUSE binding protein 1 (FUBP1), a multifunctional DNA- and RNA-binding protein whose role in splicing is largely unknown. By serial deletion and mutagenesis studies in minigenes, we delineated a functional intronic splicing enhancer (ISE) in intron 38. FUBP1 recruitment to the RNA sequence containing the ISE was established by RNA pull down and RNA EMSA, and further confirmed by RNA-ChIP on endogenous DMD pre-mRNA. This study provides new insights about the splicing regulation of DMD exon 39, highlighting the emerging role of FUBP1 in splicing and describing the first ISE for constitutive exon inclusion in the mature DMD transcript.