Complex seizure disorder caused by Brunol4 deficiency in mice

Idiopathic epilepsy is a common human disorder with a strong genetic component, usually exhibiting complex inheritance. We describe a new mouse mutation in C57BL/6J mice, called frequent-flyer (Ff), in which disruption of the gene encoding RNA-binding protein Bruno-like 4 (Brunol4) leads to limbic and severe tonic–clonic seizures in heterozygous mutants beginning in their third month. Younger heterozygous adults have a reduced seizure threshold. Although homozygotes do not survive well on the C57BL/6J background, on mixed backgrounds homozygotes and some heterozygotes also display spike-wave discharges, the electroencephalographic manifestation of absence epilepsy. Brunol4 is widely expressed in the brain with enrichment in the hippocampus. Gene expression profiling and subsequent analysis revealed the down-regulation of at least four RNA molecules encoding proteins known to be involved in neuroexcitability, particularly in mutant hippocampus. Genetic and phenotypic assessment suggests that Brunol4 deficiency in mice results in a complex seizure phenotype, likely due to the coordinate dysregulation of several molecules, providing a unique new animal model of epilepsy that mimics the complex genetic architecture of common disease.     

二色补血草LbGRP基因的克隆及抗逆能力分析

富含甘氨酸RNA结合蛋白(Glycine-rich RNA-binding proteins, GRP)是植物中重要的转录后调控蛋白, 在植物的生长发育及对胁迫的抗性调控等过程中起重要作用。文章从二色补血草(Limonium bicolor (Bunge) O.) cDNA文库中克隆出了富含甘氨酸RNA结合蛋白基因(LbGRP)的全长cDNA序列(GenBank登录号: GQ398238)。为了研究LbGRP的抗逆功能, 将其构建到酵母表达载体pYES2中, 转化到酿酒酵母INVSc1菌株中, 获得重组酵母INVSc1(pYES2-LbGRP), 同时将转空pYES2质粒的酵母INVSc1(pYES2)作为对照。对重组酵母INVSc1 (pYES2-LbGRP)和对照INVSc1(pYES2)进行NaCl、KCl、NaHCO₃、Na₂CO₃、干旱和冷冻胁迫, 比较它们在不同胁迫下的存活率。结果表明, INVSc1(pYES2-LbGRP)在各种胁迫下的存活率明显高于对照INVSc1(pYES2), 证明LbGRP基因具有抗NaCl、KCl、NaHCO₃、Na₂CO₃、干旱和冷冻等胁迫的能力, 推测该基因参与了二色补血草的抗逆调控过程。     

Alternative mRNA processing increases the complexity of microRNA-based gene regulation in Arabidopsis

During trans-splicing of discontinuous organellar introns, independently transcribed coding sequences are joined together to generate a continuous mRNA. The chloroplast psaA gene from Chlamydomonas reinhardtii encoding the P(700) core protein of photosystem I (PSI) is split into three exons and two group IIB introns, which are both spliced in trans. Using forward genetics, we isolated a novel PSI mutant, raa4, with a defect in trans-splicing of the first intron. Complementation analysis identified the affected gene encoding the 112.4 kDa Raa4 protein, which shares no strong sequence identity with other known proteins. The chloroplast localization of the protein was confirmed by confocal fluorescence microscopy, using a GFP-tagged Raa4 fusion protein. RNA-binding studies showed that Raa4 binds specifically to domains D2 and D3, but not to other conserved domains of the tripartite group II intron. Raa4 may play a role in stabilizing folding intermediates or functionally active structures of the split intron RNA.     

Structure and Expression of the Tobacco Nuclear Gene Encoding RNA-binding Protein RZ-1: The Existence of an Intron in the 3'-Untranslated Region

We have previously characterized a tobacco cDNA encoding a noveltype RNA-binding protein (RZ-1), which contains a zinc fingermotif in addition to a consensus sequence-type RNA-binding domainand is localized in the nucleus. Here we isolated its genomicclone from a Nicotiana sylvestris genomic library. Southernblot analysis suggested that RZ-1 is coded for by a single locusper haploid genome. Comparison of the cDNA and genomic sequencesindicated that the RZ-1 gene contains two introns, one in thecoding region and another in the 3'-untranslated region. RT-PCRand ribonuclease protection analyses showed that splicing ofRZ-1 pre-mRNA occurs efficiently. The RZ-1 protein is activelysynthesized in rapidly dividing tobacco cells, as demonstratedby immunoblot analysis.     

The RNA-binding protein hnRNPLL induces a T cell alternative splicing program delineated by differential intron retention in polyadenylated RNA

Background

Retention of a subset of introns in spliced polyadenylated mRNA is emerging as a frequent, unexplained finding from RNA deep sequencing in mammalian cells.

Results

Here we analyze intron retention in T lymphocytes by deep sequencing polyadenylated RNA. We show a developmentally regulated RNA-binding protein, hnRNPLL, induces retention of specific introns by sequencing RNA from T cells with an inactivating Hnrpll mutation and from B lymphocytes that physiologically downregulate Hnrpll during their differentiation. In Ptprc mRNA encoding the tyrosine phosphatase CD45, hnRNPLL induces selective retention of introns flanking exons 4 to 6; these correspond to the cassette exons containing hnRNPLL binding sites that are skipped in cells with normal, but not mutant or low, hnRNPLL. We identify similar patterns of hnRNPLL-induced differential intron retention flanking alternative exons in 14 other genes, representing novel elements of the hnRNPLL-induced splicing program in T cells. Retroviral expression of a normally spliced cDNA for one of these targets, Senp2, partially corrects the survival defect of Hnrpll-mutant T cells. We find that integrating a number of computational methods to detect genes with differentially retained introns provides a strategy to enrich for alternatively spliced exons in mammalian RNA-seq data, when complemented by RNA-seq analysis of purified cells with experimentally perturbed RNA-binding proteins.

Conclusions

Our findings demonstrate that intron retention in mRNA is induced by specific RNA-binding proteins and suggest a biological significance for this process in marking exons that are poised for alternative splicing.     

RNA结合蛋白Sam68及其功能

细胞通过基因表达调控来应对外界刺激,其中影响mRNA稳定性及翻译效率的转录后调控发挥重要作用。RNA结合蛋白(RNA binding proteins, RBPs)是介导转录后调控的重要分子,Sam68（SRC associated in mitosis of 68 kD）是集信号转导特性与RNA激活功能于一身的RNA结合蛋白,参与转录、可变剪接及核输出等mRNA 的代谢过程,且Sam68可通过信号通路参与细胞应答、细胞周期调控和疾病发生等。最新研究表明,Sam68可通过非编码RNAs(noncoding RNA, ncRNAs)参与表观遗传、转录与转录后调控。本文在介绍Sam68结构和转录后修饰的基础上,着重讨论Sam68在信号转导、可变剪接、ncRNAs代谢、疾病发生等方面的最新研究进展。