When one is better than two: RNA with dual functions

The central dogma of biology, until not long ago, held that genetic information stored on DNA molecules was translated into the final protein products through RNA as intermediate molecules. Then, an additional level of complexity in the regulation of genome expression was added, implicating new classes of RNA molecules called non-coding RNA (ncRNA). These ncRNA are also often referred to as functional RNA in that, although they do not contain the capacity to encode proteins, do have a function as RNA molecules. They have been thus far considered as truly non-coding RNA since no ORF long enough to be considered, nor protein, have been associated with them. However, the recent identification and characterization of bifunctional RNA, i.e. RNA for which both coding capacity and activity as functional RNA have been reported, suggests that a definite categorization of some RNA molecules is far from being straightforward.Indeed, several RNA primarily classified as non-protein-coding RNA has been showed to hold coding capacities and associated peptides. Conversely, mRNA, usually regarded as strictly protein-coding, may act as functional RNA molecules. Here, we describe several examples of these bifunctional RNA that have been already characterized from bacteria to mammals. We also extend this concept to fortuitous acquisition of dual function in pathological conditions and to the recently highlighted duality between information carried by a gene and its pseudogenes counterparts.     

Use of RNA interference to dissect the roles of trans-acting factors in alternative pre-mRNA splicing

RNA interference (RNAi) is becoming a popular method for analyzing gene function in a variety of biological processes. We have used RNAi in cultured Drosophila cells to identify trans-acting factors that regulate the alternative splicing of endogenously transcribed pre-mRNAs. We have generated a dsRNA library comprising 70% of the Drosophila genes encoding RNA binding proteins and assessed the function of each protein in the regulation of alternative splicing. This approach not only identifies trans-acting factors regulating specific alternative splicing events, but also can provide insight into the alternative splicing regulatory networks of Drosophila. Here, we describe this RNAi approach to identify alternative splicing regulatory proteins in detail.     

RNA在RNA剪接中的功能：从催化到调控

对非编码RNA功能的认识是后基因组时代的一个研究焦点,本文主要介绍非编码RNA在RNA剪接中的催化和调控功能。在RNA加工过程中,三大类内含子的剪接都是由RNA成员主导。其中Ⅰ型和Ⅱ型内含子能催化自身的切除和外显子连接反应;而核mRNA内含子的剪接则由剪接体里的小核RNA主导。Ⅰ型和Ⅱ型内含子存在于细菌、低等真核细胞和植物的细胞器内;而真核细胞的核编码蛋白质基因内全部是核mRNA内含子,并且其数目随生物体的复杂性而显著升高。一个多内含子前体mRNA通过选择性剪接产生多种,甚至上万种不同的mRNA和蛋白质,对蛋白质组的复杂度和时空表达调控至关重要。选择性剪接调控由剪接调控蛋白特异识别和结合前体mRNA里所富含的顺式RNA调控元件完成的;系统认识这两者之间的对应关系是揭示基因组表达调控网络的一把钥匙。     

Long-range RNA pairings contribute to mutually exclusive splicing

Mutually exclusive splicing is an important means of increasing the protein repertoire, by which the Down''s syndrome cell adhesion molecule (Dscam) gene potentially generates 38,016 different isoforms in Drosophila melanogaster. However, the regulatory mechanisms remain obscure due to the complexity of the Dscam exon cluster. Here, we reveal a molecular model for the regulation of the mutually exclusive splicing of the serpent pre-mRNA based on competition between upstream and downstream RNA pairings. Such dual RNA pairings confer fine tuning of the inclusion of alternative exons. Moreover, we demonstrate that the splicing outcome of alternative exons is mediated in relative pairing strength-correlated mode. Combined comparative genomics analysis and experimental evidence revealed similar bidirectional structural architectures in exon clusters 4 and 9 of the Dscam gene. Our findings provide a novel mechanistic framework for the regulation of mutually exclusive splicing and may offer potentially applicable insights into long-range RNA–RNA interactions in gene regulatory networks.     

Apoptotic endonuclease EndoG regulates alternative splicing of human telomerase catalytic subunit hTERT

Human telomerase catalytic subunit hTERT is subjected to alternative splicing results in loss of its function and leads to decrease of telomerase activity. However, very little is known about the mechanism of hTERT pre-mRNA alternative splicing. Apoptotic endonuclease EndoG is known to participate this process. The aim of this study was to determine the role of EndoG in regulation of hTERT alternative splicing. Increased expression of β-deletion splice variant was determined during EndoG overexpression in CaCo-2 cell line, after EndoG treatment of cell cytoplasm and nuclei as well as after nuclei incubation with EndoG digested cell RNA. hTERT alternative splicing was induced by 47-mer RNA oligonucleotide in naked nuclei and in cells after transfection. Identified long non-coding RNA, that is the precursor of 47-mer RNA oligonucleotide. Its size is 1754 nucleotides. Based on the results the following mechanism was proposed. hTERT pre-mRNA is transcribed from coding DNA strand while long non-coding RNA is transcribed from template strand of hTERT gene. EndoG digests long non-coding RNA and produces 47-mer RNA oligonucleotide complementary to hTERT pre-mRNA exon 8 and intron 8 junction place. Interaction of 47-mer RNA oligonucleotide and hTERT pre-mRNA causes alternative splicing.     

PfSR1 controls alternative splicing and steady‐state RNA levels in Plasmodium falciparum through preferential recognition of specific RNA motifs

Plasmodium species have evolved complex biology to adapt to different hosts and changing environments throughout their life cycle. Remarkably, these adaptations are achieved by a relatively small genome. One way by which the parasite expands its proteome is through alternative splicing (AS). We recently identified PfSR1 as a bona fide Ser/Arg‐rich (SR) protein that shuttles between the nucleus and cytoplasm and regulates AS in Plasmodium falciparum. Here we show that PfSR1 is localized adjacent to the Nuclear Pore Complex (NPC) clusters in the nucleus of early stage parasites. To identify the endogenous RNA targets of PfSR1, we adapted an inducible overexpression system for tagged PfSR1 and performed RNA immunoprecipitation followed by microarray analysis (RIP‐chip) to recover and identify the endogenous RNA targets that bind PfSR1. Bioinformatic analysis of these RNAs revealed common sequence motifs potentially recognized by PfSR1. RNA‐EMSAs show that PfSR1 preferentially binds RNA molecules containing these motifs. Interestingly, we find that PfSR1 not only regulates AS but also the steady‐state levels of mRNAs containing these motifs in vivo.     

Regulation of alternative RNA splicing by exon definition and exon sequences in viral and mammalian gene expression

Intron removal from a pre-mRNA by RNA splicing was once thought to be controlled mainly by intron splicing signals. However, viral and other eukaryotic RNA exon sequences have recently been found to regulate RNA splicing, polyadenylation, export, and nonsense-mediated RNA decay in addition to their coding function. Regulation of alternative RNA splicing by exon sequences is largely attributable to the presence of two majorcis-acting elements in the regulated exons, the exonic splicing enhancer (ESE) and the suppressor or silencer (ESS). Two types of ESEs have been verified from more than 50 genes or exons: purine-rich ESEs, which are the more common, and non-purine-rich ESEs. In contrast, the sequences of ESSs identified in approximately 20 genes or exons are highly diverse and show little similarity to each other. Through interactions with cellular splicing factors, an ESE or ESS determines whether or not a regulated splice site, usually an upstream 3 splice site, will be used for RNA splicing. However, how these elements function precisely in selecting a regulated splice site is only partially understood. The balance between positive and negative regulation of splice site selection likely depends on thecis-element's identity and changes in cellular splicing factors under physiological or pathological conditions.     

Redirecting splicing with bifunctional oligonucleotides

Ectopic modulators of alternative splicing are important tools to study the function of splice variants and for correcting mis-splicing events that cause human diseases. Such modulators can be bifunctional oligonucleotides made of an antisense portion that determines target specificity, and a non-hybridizing tail that recruits proteins or RNA/protein complexes that affect splice site selection (TOSS and TOES, respectively, for targeted oligonucleotide silencer of splicing and targeted oligonucleotide enhancer of splicing). The use of TOSS and TOES has been restricted to a handful of targets. To generalize the applicability and demonstrate the robustness of TOSS, we have tested this approach on more than 50 alternative splicing events. Moreover, we have developed an algorithm that can design active TOSS with a success rate of 80%. To produce bifunctional oligonucleotides capable of stimulating splicing, we built on the observation that binding sites for TDP-43 can stimulate splicing and improve U1 snRNP binding when inserted downstream from 5′ splice sites. A TOES designed to recruit TDP-43 improved exon 7 inclusion in SMN2. Overall, our study shows that bifunctional oligonucleotides can redirect splicing on a variety of genes, justifying their inclusion in the molecular arsenal that aims to alter the production of splice variants.     

非编码RNA调控血管平滑肌细胞成骨样表型转化的研究进展

分化成熟的血管平滑肌主要功能是收缩血管、调节血管周径及血压等.在高磷、高糖、维生素D₃、炎症等因素的作用下,平滑肌细胞可转分化为成骨样细胞参与血管钙化的形成,诱发心脑血管不良事件.非编码RNA是经基因转录但不翻译为蛋白质的一类RNA总称,其通过调控多种细胞活动来参与机体的生理和病理过程.已有研究表明,非编码RNA可通过调控血管平滑肌细胞成骨样表型转化影响血管钙化的发生、发展.本文从微小RNA、长链非编码RNA、环状RNA几方面综述非编码RNA在血管平滑肌成骨样表型转化中的调节作用,有助于进一步了解血管钙化的分子机制以及发现防治血管钙化的新靶点.     

The yeast mitochondrial leucyl-tRNA synthetase is a splicing factor for the excision of several group I introns

Summary The Saccharomyces cerevisiae nuclear gene NAM2 codes for mitochondrial leucyl-tRNA synthetase (mLRS). Herbert et al. (1988, EMBO J 7:473–483) proposed that this protein is involved in mitochondrial RNA splicing. Here we present the construction and analyses of nine mutations obtained by creating two-codon insertions within the NAM2 gene. Three of these prevent respiration while maintaining the mitochondrial genome. These three mutants: (1) display in vitro a mLRS activity ranging from 0%–50% that of the wild type: (2) allow in vivo the synthesis of several mitochondrially encoded proteins; (3) prevent the synthesis of the COXII protein but not of its mRNA; (4) abolish the splicing of the group I introns bI4 and aI4; and (5) affect significantly the excision of the group I introns bI2, bI3 and aI3. Importation of the bI4 maturase from the cytoplasm into mitochondria in a nam2 ^– mutant strain does not restore the excision of the introns bI4 and aI4 implying that the splicing deficiency does not result from the absence of the bI4 maturase. We conclude that the mLRS is a splicing factor essential for the excision of the group I introns bI4 and aI4 and probably important for the excision of other group I introns.     

Regulation of protein diversity by alternative pre-mRNA splicing with specific focus on chondrogenesis

Analysis of the human genome has dramatically demonstrated that the majority of protein diversity is generated by alternative splicing of pre-mRNA. This powerful and versatile mechanism controls the synthesis of functionally different protein isoforms that may be required during specific stages of development from a single gene. Consequently, ubiquitous and/or tissue-specific RNA splicing factors that regulate this splicing mechanism provide the basis for defining phenotypic characteristics of cells during differentiation. In this review, we will introduce the basic mechanisms of pre-mRNA alternative splicing, describe how this process is regulated by specific RNA splicing factors, and relate this to various systems of cell differentiation. Chondrogenesis, a well-defined differentiation pathway necessary for skeletogenesis, will be discussed in detail, with focus on some of the alternatively-spliced proteins known to be expressed during cartilage development. We propose a heuristic view that, ultimately, it is the regulation of these RNA splicing factors that determines the differentiation status of a cell. Studying regulation at the level of pre-mRNA alternative splicing will provide invaluable insights into how many developmental mechanisms are controlled, thus enabling us to manipulate a system to select for a specific differentiation pathway.     

YTH Domain: A Family of N6-methyladenosine (m6A) Readers

Like protein and DNA, different types of RNA molecules undergo various modifications. Accumulating evidence suggests that these RNA modifications serve as sophisticated codes to mediate RNA behaviors and many important biological functions. N⁶-methyladenosine (m⁶A) is the most abundant internal RNA modification found in a variety of eukaryotic RNAs, including but not limited to mRNAs, tRNAs, rRNAs, and long non-coding RNAs (lncRNAs). In mammalian cells, m⁶A can be incorporated by a methyltransferase complex and removed by demethylases, which ensures that the m⁶A modification is reversible and dynamic. Moreover, m⁶A is recognized by the YT521-B homology (YTH) domain-containing proteins, which subsequently direct different complexes to regulate RNA signaling pathways, such as RNA metabolism, RNA splicing, RNA folding, and protein translation. Herein, we summarize the recent progresses made in understanding the molecular mechanisms underlying the m⁶A recognition by YTH domain-containing proteins, which would shed new light on m⁶A-specific recognition and provide clues to the future identification of reader proteins of many other RNA modifications.