RBM22 regulates RNA polymerase II 5' pausing, elongation rate, and termination by coordinating 7SK-P-TEFb complex and SPT5.

BACKGROUND: Splicing factors are vital for the regulation of RNA splicing, but some have also been implicated in regulating transcription. The underlying molecular mechanisms of their involvement in transcriptional processes remain poorly understood. RESULTS: Here, we describe a direct role of splicing factor RBM22 in coordinating multiple steps of RNA Polymerase II (RNAPII) transcription in human cells. The RBM22 protein widely occupies the RNAPII-transcribed gene locus in the nucleus. Loss of RBM22 promotes RNAPII pause release, reduces elongation velocity, and provokes transcriptional readthrough genome-wide, coupled with production of transcripts containing sequences from downstream of the gene. RBM22 preferentially binds to the hyperphosphorylated, transcriptionally engaged RNAPII and coordinates its dynamics by regulating the homeostasis of the 7SK-P-TEFb complex and the association between RNAPII and SPT5 at the chromatin level. CONCLUSIONS: Our results uncover the multifaceted role of RBM22 in orchestrating the transcriptional program of RNAPII and provide evidence implicating a splicing factor in both RNAPII elongation kinetics and termination control.

Pervasive Chromatin-RNA Binding Protein Interactions Enable RNA-Based Regulation of Transcription.

Increasing evidence suggests that transcriptional control and chromatin activities at large involve regulatory RNAs, which likely enlist specific RNA-binding proteins (RBPs). Although multiple RBPs have been implicated in transcription control, it has remained unclear how extensively RBPs directly act on chromatin. We embarked on a large-scale RBP ChIP-seq analysis, revealing widespread RBP presence in active chromatin regions in the human genome. Like transcription factors (TFs), RBPs also show strong preference for hotspots in the genome, particularly gene promoters, where their association is frequently linked to transcriptional output. Unsupervised clustering reveals extensive co-association between TFs and RBPs, as exemplified by YY1, a known RNA-dependent TF, and RBM25, an RBP involved in splicing regulation. Remarkably, RBM25 depletion attenuates all YY1-dependent activities, including chromatin binding, DNA looping, and transcription. We propose that various RBPs may enhance network interaction through harnessing regulatory RNAs to control transcription.

A new Kunitz-type plasmin inhibitor from scorpion venom.

Kunitz-type peptides from venomous animals are an important source of lead drug candidates towards human plasmin, a target of protease-associated diseases. However, no Kunitz-type plasmin inhibitor from venomous scorpion has been characterized. Here, we first investigated plasmin inhibiting activities of eight known Kunitz-type scorpion toxins Hg1, BmKTT-1, BmKTT-2, BmKTT-3, LmKTT-1a, LmKTT-1b, LmKTT-1c and BmKPI, and found a new plasmin inhibitor BmKTT-2, a Kunitz-type toxin peptide from the scorpion Buthus martensi karch. Protease inhibitory activity assay showed that BmKTT-2 potently inhibited plasmin with a Ki value of 8.75 ± 2.05 nM. Structure-function relationship studies between BmKTT-2 and plasmin showed that BmKTT-2 is a classical Kunitz-type plasmin inhibitor: Lys13 in BmKTT-2 is the P1 site, and Ala14 in BmKTT-2 is the P1' site. Interestingly, BmKTT-2 has potent inhibiting activities towards three important digestive serine proteases trypsin, chymotrypsin and elastase, suggesting a good stability for administering oral medications. To the best of our knowledge, BmKTT-2 is the first Kunitz-type human plasmin inhibitor from scorpion venom, providing novel insights into drug developments targeting human plasmin protease.

Cloning and Genomic Characterization of a Natural Insecticidal Peptide LaIT1 with Unique DDH Structural Fold.

Two native peptides with disulfide-directed hairpin (DDH) fold, LaIT1 and LITX, were recently isolated from scorpion venom, a development that offered insights into exploring the evolutionary linkage between DDH and inhibitor cystine knot (ICK) peptides. In this work, we isolated and identified the full-length cDNAs of LaTI1, a representative member with DDH fold, and further determined its complete gene structure. The precursor organization of LaIT1 is similar to that of ICK peptides. The LaIT1 gene contains four exons interrupted by three unique introns and differed from ICK peptides, suggesting divergent genomic organizations of DDH peptides and ICK peptides. Phylogenetic analysis further showed that the "simple" DDH peptide originates from the "complex" ICK peptide, rather than the reverse. To the best of our knowledge, this is the first report on the genomic organization of DDH-fold peptides, and it presents new evidence of an evolutionary linkage between ICK and DDH peptides.

Functional characterization of a new non-Kunitz serine protease inhibitor from the scorpion Lychas mucronatus.

Serine protease inhibitors have been widely discovered from different animal venoms, but most of them belong to Kunitz-type toxin subfamily. Here, by screening scorpion venom gland cDNA libraries, we identified four new non-Kunitz serine protease inhibitors with a conserved Ascaris-type structural fold: Ascaris-type toxins Lychas mucronatus Ascaris-type protease inhibitor (LmAPI), Pandinus cavimanus Ascaris-type protease inhibitor (PcAPI), Pandinus cavimanus Ascaris-type protease inhibitor 2 (PcAPI-2), and Hottentotta judaicus Ascaris-type protease inhibitor (HjAPI). The detailed characterization of one Ascaris-type toxin LmAPI was further carried out, which contains 60 residues and possesses a classical Ascaris-type cysteine framework reticulated by five disulfide bridges. Enzyme and inhibitor reaction kinetics experiments showed that recombinant LmAPI inhibits the activity of chymotrypsin potently with a Ki value of 15.5 nM, but has little effect on trypsin and elastase. Bioinformatics analyses suggested that LmAPI contains unique functional residues "TQD" and might be a useful template to produce specific protease inhibitors. Our results indicated that animal venoms are a natural source of new type of protease inhibitors, which will accelerate the development of diagnostic and therapeutic agents for human diseases that target diverse proteases.