Structural architecture of the human long non-coding RNA, steroid receptor RNA activator

While functional roles of several long non-coding RNAs (lncRNAs) have been determined, the molecular mechanisms are not well understood. Here, we report the first experimentally derived secondary structure of a human lncRNA, the steroid receptor RNA activator (SRA), 0.87 kB in size. The SRA RNA is a non-coding RNA that coactivates several human sex hormone receptors and is strongly associated with breast cancer. Coding isoforms of SRA are also expressed to produce proteins, making the SRA gene a unique bifunctional system. Our experimental findings (SHAPE, in-line, DMS and RNase V1 probing) reveal that this lncRNA has a complex structural organization, consisting of four domains, with a variety of secondary structure elements. We examine the coevolution of the SRA gene at the RNA structure and protein structure levels using comparative sequence analysis across vertebrates. Rapid evolutionary stabilization of RNA structure, combined with frame-disrupting mutations in conserved regions, suggests that evolutionary pressure preserves the RNA structural core rather than its translational product. We perform similar experiments on alternatively spliced SRA isoforms to assess their structural features.     

Cell type specific trans-acting factors are involved in alternative splicing of human fibronectin pre-mRNA.

ED-A and ED-B are facultative type III homologies of fibronectin, encoded by alternatively spliced exons, described in man and in rat. A hybrid alpha-globin-fibronectin minigene containing the ED-B region from the human gene has been transfected in human cell lines derived from various tissues, in order to study the processing of the generated precursor RNA in the different cell environments. In most tested lines the pre-RNA is alternatively spliced and produces two mature RNAs, with and without the ED-B exon, in different ratios that closely resemble the corresponding endogenous fibronectin RNAs. In a hepatoma cell line, Hep 3B, only one RNA is produced, in which the ED-B exon is absent; the same pattern of splicing is observed in liver. The data show that all the information required to produce accurate and regulated alternative splicing of the ED-B exon is contained in the fragment used and cell specific factors are necessary for the pre-RNA to be differentially spliced in the various cell lines. In contrast, expression in Hep 3B of a similar gene containing the ED-A area failed to reproduce the liver specific splicing pattern. Therefore regulation of ED-A processing is likely to involve different mechanisms to those responsible for control of ED-B splicing.     

Alternative splicing of human immunodeficiency virus type 1 mRNA modulates viral protein expression, replication, and infectivity.

Multiple RNA splicing sites exist within human immunodeficiency virus type 1 (HIV-1) genomic RNA, and these sites enable the synthesis of many mRNAs for each of several viral proteins. We evaluated the biological significance of the alternatively spliced mRNA species during productive HIV-1 infections of peripheral blood lymphocytes and human T-cell lines to determine the potential role of alternative RNA splicing in the regulation of HIV-1 replication and infection. First, we used a semiquantitative polymerase chain reaction of cDNAs that were radiolabeled for gel analysis to determine the relative abundance of the diverse array of alternatively spliced HIV-1 mRNAs. The predominant rev, tat, vpr, and env RNAs contained a minimum of noncoding sequence, but the predominant nef mRNAs were incompletely spliced and invariably included noncoding exons. Second, the effect of altered RNA processing was measured following mutagenesis of the major 5' splice donor and several cryptic, constitutive, and competing 3' splice acceptor motifs of HIV-1NL4-3. Mutations that ablated constitutive splice sites led to the activation of new cryptic sites; some of these preserved biological function. Mutations that ablated competing splice acceptor sites caused marked alterations in the pool of virus-derived mRNAs and, in some instances, in virus infectivity and/or the profile of virus proteins. The redundant RNA splicing signals in the HIV-1 genome and alternatively spliced mRNAs provides a mechanism for regulating the relative proportions of HIV-1 proteins and, in some cases, viral infectivity.     

Assembly in an in vitro splicing reaction of a mouse insulin messenger RNA precursor into a 60-40S ribonucleoprotein complex.

An SP6/mouse insulin RNA precursor containing two exons and one intron can be spliced in a partially purified nuclear extract isolated from MOPC-315 mouse myeloma cells. We have detected the putative RNA splicing intermediate (intron-3'exon) in a lariat form, the excised intron in a lariat form, and the mRNA spliced product. The in vitro splicing reaction of gel-purified RNA precursors requires ATP and Mg2+ and was accompanied by the formation of a 60-40S ribonucleoprotein complex. The formation of the 60S complex requires ATP. At least two Sm snRNPs containing U1 and U2 RNAs are components of the 60-40S complex. The assemble of those snRNPs occurs early during the splicing reaction and it requires ATP and intron containing pre-mRNAs.     

SRA的RNA和蛋白质均有转录辅激活功能

类固醇受体激活物（steroid receptor activator, SRA）是一种 类固醇受体辅激活物.最初的研究认为,SRA只存在RNA形式,不存在蛋白质 形式.但是后来的研究发现,SRA是在RNA和蛋白质两个水平上发挥功能的分 子,其cDNA序列存在687 bp保守的核心区域,该核心区域对其发挥转录共激 活活性是必需的.SRA的RNA形式主要参与核受体的转录共激活作用,其表达 与乳腺癌的发生有很大关系,SRA的蛋白质形式（steroid receptor activator protein ,SRAP）也具有类似的功能.但是不同于RNA形式, SRAP可结合到特定基因的启动子区域,并起到阻遏物的作用.本文对SRA的 特点、表达及功能等方面的最新研究进展及其可能的作用机制与作用形式 进行概述.     

RNA binding protein RALY promotes Protein Arginine Methyltransferase 1 alternatively spliced isoform v2 relative expression and metastatic potential in breast cancer cells

Aberrant expression of Protein Arginine Methyltransferases (PRMTs) has been observed in several cancer types, including breast cancer. We previously reported that the PRMT1v2 isoform, which is generated through inclusion of alternative exon 2, is overexpressed in breast cancer cells and promotes their invasiveness. However, the precise mechanism by which expression of this isoform is controlled and how it is dysregulated in breast cancer remains unknown. Using a custom RNA interference-based screen, we identified several RNA binding proteins (RBP) which, when knocked down, altered the relative abundance of the alternatively spliced PRMT1v2 isoform. Amongst the top hits were SNW Domain containing 1 (SNW1) and RBP-associated with lethal yellow mutation (RALY), which both associated with the PRMT1 pre-mRNA and upon depletion caused an increase or decrease in the relative abundance of PRMT1v2 isoform mRNA and protein. Most importantly, a significant decrease in invasion was observed upon RALY knockdown in aggressive breast cancer cells, consistent with targeting PRMT1v2 directly, and this effect was rescued by the exogenous re-expression of PRMT1v2. We show that SNW1 expression is decreased, while RALY expression is increased in breast cancer cells and tumours, which correlates with decreased patient survival. This work revealed crucial insight into the mechanisms regulating the expression of the PRMT1 alternatively spliced isoform v2 and its dysregulation in breast cancer. It also provides proof-of-concept support for the development of therapeutic strategies where regulators of PRMT1 exon 2 alternative splicing are targeted as an approach to selectively reduce PRMT1v2 levels and metastasis in breast cancer.     

Trans-complementation of the second step of pre-mRNA splicing by exogenous 5' exons.

During splicing of nuclear pre-mRNAs, the first step liberates the 5' exon (exon 1) and yields a lariat intron-3'exon (intron-exon 2) intermediate. The second step results in exon ligation. Previous results indicated that severe truncations of the 5' exon of the actin pre-mRNA result in a block to the second splicing step in vitro in yeast extracts, leading to an accumulation of intron-exon 2 lariat intermediates. We show that exogenous exon 1 RNA oligonucleotides can chase these stalled intermediates into lariat intron and spliced exons. This reaction requires some of the cis elements and trans-acting factors that are required for a normal second step. There is no strong sequence requirement for the exon 1 added in trans, but oligonucleotides with complementarity to the U5 snRNA conserved loop perform the chase more efficiently. Using a dominant negative mutant of the DEAH-box ATPase Prp16p and ATP depletion, we show that the stalled intermediate is blocked after the Prp16p-dependent step. These results show that exogenous RNAs with various sequences but containing no splicing signals can be incorporated into spliceosomes and undergo RNA recombination and exon shuffling during the second step of pre-mRNA splicing.     

Identification of an hnRNP A1-dependent splicing silencer in the human papillomavirus type 16 L1 coding region that prevents premature expression of the late L1 gene

We have previously identified cis-acting RNA sequences in the human papillomavirus type 16 (HPV-16) L1 coding region which inhibit expression of L1 from eukaryotic expression plasmids. Here we have determined the function of one of these RNA elements, and we provide evidence that this RNA element is a splicing silencer which suppresses the use of the 3' splice site located immediately upstream of the L1 AUG. We also show that this splice site is inefficiently utilized as a result of a suboptimal polypyrimidine tract. Introduction of point mutations in the L1 coding region that altered the RNA sequence without affecting the L1 protein sequence resulted in the inactivation of the splicing silencer and induced splicing to the L1 3' splice site. These mutations also prevented the interaction of the RNA silencer with a 35-kDa cellular protein identified here as hnRNP A1. The splicing silencer in L1 inhibits splicing in vitro, and splicing can be restored by the addition of RNAs containing an hnRNP A1 binding site to the reaction, demonstrating that hnRNP A1 inhibits splicing of the late HPV-16 mRNAs through the splicing silencer sequence. While we show that one role of the splicing silencer is to determine the ratio between partially spliced L2/L1 mRNAs and spliced L1 mRNAs, we also demonstrate that it inhibits splicing from the major 5' splice site in the early region to the L1 3' splice site, thereby playing an essential role in preventing late gene expression at an early stage of the viral life cycle. We speculate that the activity of the splicing silencer and possibly the concentration of hnRNP A1 in the HPV-16-infected cell determines the ability of the virus to establish a persistent infection which remains undetected by the host immune surveillance.