Eukaryotic Post transcriptional Gene Regulation

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The splicing factor ASF/SF2 is associated with TIA-1-related/ TIA-1-containing ribonucleoproteic complexes and contributes to post-transcriptional repression of gene expression Nathalie Delestienne 1 , Corinne Wauquier 1 , Romuald Soin 1 , Jean-Franc¸ois Dierick 2, *, Cyril Gueydan 1, and Ve ´ ronique Kruys 1, 1 Laboratoire de Biologie Mole ´ culaire du Ge ` ne, Faculte ´ des Sciences, Universite ´ Libre de Bruxelles, Gosselies, Belgium 2 Biovalle ´ e, Proteomics Unit, Charleroi, Belgium Keywords AU-rich elements; hnRNP, heterogenous nuclear ribonucleoprotein; ribonucleoprotein complexes; RNA metabolism; RNA-binding proteins; stress granules Correspondence V. Kruys, Laboratoire de Biologie Mole ´ culaire du Ge ` ne, Institut de Biologie et de Me ´ decine Mole ´ culaires, Universite ´ Libre de Bruxelles, 12 rue des Profs. Jeener et Brachet, 6041 Gosselies, Belgium Fax: +32 2 6509800 Tel: +32 2 6509804 E-mail: vkruys@ulb.ac.be *Present address GSK Biologicals, Wavre, Belgium  These authors contributed equally to this work (Received 10 January 2010, revised 10 March 2010, accepted 25 March 2010) doi:10.1111/j.1742-4658.2010.07664.x TIA-1-related (TIAR) protein is a shuttling RNA-binding protein impli- cated in several steps of RNA metabolism. In the nucleus, TIAR contrib- utes to alternative splicing events, whereas, in the cytoplasm, it acts as a translational repressor on specific transcripts such as adenine and uridine- rich element-containing mRNAs. In addition, TIAR is involved in the general translational arrest observed in cells exposed to environmental stress. This activity is encountered by the ability of TIAR to assemble abortive pre-initiation complexes coalescing into cytoplasmic granules called stress granules. To elucidate these mechanisms of translational repression, we characterized TIAR-containing complexes by tandem affinity purification followed by MS. Amongst the identified proteins, we found the splicing factor ASF ⁄ SF2, which is also present in TIA-1 protein complexes. We show that, although mostly confined in the nuclei of normal cells, ASF ⁄ SF2 migrates into stress granules upon environmental stress. The migration of ASF ⁄ SF2 into stress granules is strictly determined both by its shuttling properties and its RNA-binding capacity. Our data also indi- cate that ASF ⁄ SF2 down-regulates the expression of a reporter mRNA carrying adenine and uridine-rich elements within its 3¢ UTR. Moreover, tethering of ASF ⁄ SF2 to a reporter transcript strongly reduces mRNA translation and stability. These results indicate that ASF ⁄ SF2 and TIA proteins cooperate in the regulation of mRNA metabolism in normal cells and in cells having to overcome environmental stress conditions. In addi- tion, the present study provides new insights into the cytoplasmic function of ASF ⁄ SF2 and highlights mechanisms by which RNA-binding proteins regulate the diverse steps of RNA metabolism by subcellular relocalization upon extracellular stimuli. Structured digital abstract l MINT-7715509: ASF ⁄ SF2 (uniprotkb:Q6PDM2)andTIAR (uniprotkb:P70318) colocalize (MI:0403) by fluorescence microscopy ( MI:0416) Abbreviations ARE, adenine and uridine-rich element; CBB, calmodulin binding buffer; CP, coat protein; FITC, fluorescein isothiocyanate; Fluc, firefly luciferase; HA, haemagglutinin; IP, immunoprecipitation; NLS, nuclear localization signal; NPc, nucleoplasmin core domain; Rluc, Renilla luciferase; RRM, RNA recognition motif; RS, arginine-serine; SG, stress granule; SR, serine-arginine; TAP, tandem affinity purification; TIAR, TIA-1-related. 2496 FEBS Journal 277 Eukaryotic Post-transcriptional Gene Regulation Eukaryotic Posttranscriptional Gene Regulation Bởi: OpenStaxCollege RNA is transcribed, but must be processed into a mature form before translation can begin This processing after an RNA molecule has been transcribed, but before it is translated into a protein, is called post-transcriptional modification As with the epigenetic and transcriptional stages of processing, this post-transcriptional step can also be regulated to control gene expression in the cell If the RNA is not processed, shuttled, or translated, then no protein will be synthesized RNA splicing, the first stage of post-transcriptional control In eukaryotic cells, the RNA transcript often contains regions, called introns, that are removed prior to translation The regions of RNA that code for protein are called exons ([link]) After an RNA molecule has been transcribed, but prior to its departure from the nucleus to be translated, the RNA is processed and the introns are removed by splicing Pre-mRNA can be alternatively spliced to create different proteins Evolution Connection Alternative RNA SplicingIn the 1970s, genes were first observed that exhibited alternative RNA splicing Alternative RNA splicing is a mechanism that allows different protein products to be produced from one gene when different combinations of introns, and sometimes exons, are removed from the transcript ([link]) This alternative splicing can be haphazard, but more often it is controlled and acts as a mechanism of gene 1/5 Eukaryotic Post-transcriptional Gene Regulation regulation, with the frequency of different splicing alternatives controlled by the cell as a way to control the production of different protein products in different cells or at different stages of development Alternative splicing is now understood to be a common mechanism of gene regulation in eukaryotes; according to one estimate, 70 percent of genes in humans are expressed as multiple proteins through alternative splicing There are five basic modes of alternative splicing How could alternative splicing evolve? Introns have a beginning and ending recognition sequence; it is easy to imagine the failure of the splicing mechanism to identify the end of an intron and instead find the end of the next intron, thus removing two introns and the intervening exon In fact, there are mechanisms in place to prevent such intron skipping, but mutations are likely to lead to their failure Such “mistakes” would more than likely produce a nonfunctional protein Indeed, the cause of many genetic diseases is alternative splicing rather than mutations in a sequence However, alternative splicing would create a protein variant without the loss of the original protein, opening up possibilities for adaptation of the new variant to new functions Gene duplication has played an important role in the evolution of new functions in a similar way by providing genes that may evolve without eliminating the original, functional protein Link to Learning 2/5 Eukaryotic Post-transcriptional Gene Regulation Visualize how mRNA splicing happens by watching the process in action in this video Control of RNA Stability Before the mRNA leaves the nucleus, it is given two protective "caps" that prevent the end of the strand from degrading during its journey The 5' cap, which is placed on the 5' end of the mRNA, is usually composed of a methylated guanosine triphosphate molecule (GTP) The poly-A tail, which is attached to the 3' end, is usually composed of a series of adenine nucleotides Once the RNA is transported to the cytoplasm, the length of time that the RNA resides there can be controlled Each RNA molecule has a defined lifespan and decays at a specific rate This rate of decay can influence how much protein is in the cell If the decay rate is increased, the RNA will not exist in the cytoplasm as long, shortening the time for translation to occur Conversely, if the rate of decay is decreased, the RNA molecule will reside in the cytoplasm longer and more protein can be translated This rate of decay is referred to as the RNA stability If the RNA is stable, it will be detected for longer periods of time in the cytoplasm Binding of proteins to the RNA can influence its stability Proteins, called RNA-binding proteins, or RBPs, can bind to the regions of the RNA just upstream or downstream of the protein-coding region These regions in the RNA that are not translated into protein are called the untranslated regions, or UTRs They are not introns (those have been removed in the nucleus) Rather, these are regions that regulate mRNA localization, stability, and protein translation The region just before the protein-coding region is called the 5' UTR, whereas the region after the coding region is called the 3' UTR ([link]) The binding of RBPs to these regions can increase or decrease the stability of an RNA molecule, depending on the specific RBP that binds The protein-coding region of mRNA is flanked by 5' and ...MINIREVIEW The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression Christophe Maris*, Cyril Dominguez* and Fre ´ de ´ ric H T. Allain Institute for Molecular Biology and Biophysics, Swiss Federal Institute of Technology Zurich, ETH-Ho ¨ nggerberg, Zu ¨ rich, Switzerland History – what defines an RRM? The RNA recognition motif (RRM), also known as the RNA-binding domain (RBD) or ribonucleopro- tein domain (RNP), was first identified in the late 1980s when it was demonstrated that mRNA precur- sors (pre-mRNA) and heterogeneous nuclear RNAs (hnRNAs) are always found in complex with proteins (reviewed in [1]). Biochemical characterizations of the mRNA polyadenylate binding protein (PABP) and the hnRNP protein C shed light on a consensus RNA-binding domain of approximately 90 amino acids containing a central sequence of eight con- served residues that are mainly aromatic and posi- tively charged [2,3]. This sequence, termed the RNP consensus sequence, was thought to be involved in RNA interaction and was defined as Lys ⁄ Arg- Gly-Phe ⁄ Tyr-Gly ⁄ Ala-Phe ⁄ Tyr-Val ⁄ Ile ⁄ Leu-X-Phe ⁄ Tyr, where X can be any amino acid. Later, a second consensus sequence less conserved than the previously characterized one [1] was identified. This six residue sequence located at the N-terminus of the domain Keywords RNA recognition motif; protein–RNA complex; structure–function relationship; RNA-binding specificity Correspondence F. H T. Allain, Institute for Molecular Biology and Biophysics, Swiss Federal Institute of Technology Zurich, ETH- Ho ¨ nggerberg, CH-8093 Zu ¨ rich, Switzerland Fax: +41 1 6331294 Tel: +41 1 6333940 E-mail: allain@mol.biol.ethz.ch Website: http://www.mol.biol.ethz.ch/ groups/allain_group *These authors contributed equally to the work (Received 16 December 2004, accepted 7 March 2005) doi:10.1111/j.1742-4658.2005.04653.x The RNA recognition motif (RRM), also known as RNA-binding domain (RBD) or ribonucleoprotein domain (RNP) is one of the most abundant protein domains in eukaryotes. Based on the comparison of more than 40 structures including 15 complexes (RRM–RNA or RRM–protein), we reviewed the structure–function relationships of this domain. We identified and classified the different structural elements of the RRM that are import- ant for binding a multitude of RNA sequences and proteins. Common structural aspects were extracted that allowed us to define a structural leit- motif of the RRM–nucleic acid interface with its variations. Outside of the two conserved RNP motifs that lie in the center of the RRM b-sheet, the two external b-strands, the loops, the C- and N-termini, or even a second RRM domain allow high RNA-binding affinity and specific recognition. Protein–RRM interactions that have been found in several structures rein- force the notion of an extreme structural versatility of this domain support- ing the numerous biological functions of the RRM-containing proteins. Abbreviations ACF, APOBEC-1 complementary factor; CBP, cap binding protein; CstF, cleavage stimulation factor; hnRNP, heterogeneous nuclear ribonucleoprotein; HuD, Hu protein D; LRR, leucine rich repeat; MIF4G, middle domain of the translation initiation factor 4 G; PABP, polyadenylate binding protein; PIE, polyadenylation inhibition element; PTB, polypyrimidine tract binding protein; RBD, RNA-binding domain; RNP, ribonucleoprotein; RRM, RNA recognition motif; SR, serine/arginine rich proteins; TLS, translocated in liposarcoma; U1A, U2A¢, U2B¢: U1 snRNP proteins A, A¢,B¢; U2AF, U2 snRNP auxiliary factor; UHM, U2AF homology motif; UPF, up-frameshift protein. 2118 FEBS Journal Loss of sense transgene-induced post-transcriptional gene silencing by sequential introduction of the same transgene sequences in tobacco Sayaka Hirai 1 , Kouta Takahashi 2 , Tomomi Abiko 3 and Hiroaki Kodama 1 1 Graduate School of Horticulture, Chiba University, Japan 2 Graduate School of Science and Technology, Chiba University, Japan 3 Faculty of Horticulture, Chiba University, Japan Introduction Genetic transformation is a powerful tool of improve- ment of plant physiological traits, and is important to both basic and applied sciences. Successful expression of transgene sequences is always desired, and overex- pression of the transgene is usually selected in a popu- lation of transgenic plants. The transgene itself is often recognized as a sequence of invasive nucleic acids and triggers RNA silencing [1–4]. RNA silencing, an RNA- mediated suppression of gene activity, is a common phenomenon in most eukaryotic organisms. Sense transgene-induced post-transcriptional gene silencing (S-PTGS) is a representative phenomenon of RNA silencing targeting the sense transgene, and the transgene and its homologous endogenous genes are suppressed simultaneously [5,6]. S-PTGS is usually observed in a portion of transgenic plants. In S-PTGS Keywords fatty acid desaturase; post-transcriptional gene silencing; RNA-directed DNA methylation; threshold; a-linolenic acid Correspondence H. Kodama, Graduate School of Horticulture, Chiba University, 648 Matsudo, Chiba 271-8510, Japan Fax: +81 43 290 3942 Tel: +81 43 290 3942 E-mail: kodama@faculty.chiba-u.jp (Received 31 October 2009, revised 23 January 2010, accepted 26 January 2010) doi:10.1111/j.1742-4658.2010.07591.x RNA silencing is an epigenetic inhibition of gene expression and is guided by small interfering RNAs. Sense transgene-induced post-transcriptional gene silencing (S-PTGS) occurs in a portion of a transgenic plant popula- tion. When a sense transgene encoding a tobacco endoplasmic reticulum x-3 fatty acid desaturase (NtFAD3) was introduced into tobacco plants, an S-PTGS line, S44, was obtained. Introduction of another copy of the NtFAD3 transgene into S44 plants caused a phenotypic change from S-PTGS to overexpression. Because this change was associated with the methylation of the promoter sequences of the transgene, reduced transcrip- tional activity may abolish S-PTGS and residual transcription of the sense transgene may account for the overexpression. To clarify whether RNA-directed DNA methylation (RdDM) can repress the transcriptional activity of the S44 transgene locus, we introduced several RdDM constructs targeting the transgene promoter. An RdDM construct harbor- ing a 200-bp-long fragment of promoter sequences efficiently abrogated the generation of NtFAD3 small interfering RNAs in S44 plants. Transcription of the transgene was partially repressed, but the resulting NtFAD3 mRNAs successfully accumulated and an overexpressed phenotype was established. Our results indicate an example in which overexpression of the transgene is established by complex epigenetic interactions among the transgenic loci. Abbreviations CaMV, cauliflower mosaic virus; ChIP, chromatin immunoprecipitation; CHS, chalcone synthase gene; GFP, green fluorescent protein; GUS, b-glucuronidase; H3K4me3, histone H3 trimethylated at lysine-4; H3K9me2, histone H3 dimethylated at lysine-9; Nii, tobacco nitrite reductase gene; Nos, REVIEW ARTICLE Functional interplay between viral and cellular SR proteins in control of post-transcriptional gene regulation Ming-Chih Lai 1, *, Tsui-Yi Peng 1,2, * and Woan-Yuh Tarn 1 1 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 2 Institute of Molecular Medicine, National Tsing Hua University, Hsin-Chu, Taiwan Introduction Arginine ⁄ serine (RS) dipeptide repeats are present in a number of cellular proteins, termed SR proteins, that primarily participate in nuclear precursor (pre)-mRNA splicing [1–3]. RS domain variants, such as serine and arginine-rich motifs or arginine–aspartate or arginine– glutamate dipeptide-rich domains, are also found in many nuclear proteins. In addition to the RS domains, SR splicing factors often contain one or more RNA recognition motifs. SR proteins function in both constitutive and regulated splicing via binding to cis-elements of pre-mRNA or interaction with other splicing factors. The RS domain interacts with both proteins and RNAs [1–3]. In particular, intermolecular interactions between SR proteins, which are important for spliceosome assembly and splice site determination during pre-mRNA splicing, are mediated by their RS domains [3]. The RS domain also acts as a nuclear localization signal and targets SR proteins to nuclear speckled domains, where splicing factors are concen- trated, for storage [1]. An important biochemical property of the RS domain is its differential phosphorylation at multiple serine and threonine residues. The RS domain is primarily phos- phorylated by SR protein-specific kinases (SRPKs), and Keywords Alternative splicing; kinases; phosphatases; phosphorylation; post-transcriptional control; pre-mRNA splicing; RS domain; SR proteins; viral problems; virus Correspondence W Y. Tarn, Institute of Biomedical Sciences, Academia Sinica, 128 Academy Road, Section 2, Nankang, Taipei 11529, Taiwan Fax: +886 2 2782 9142 Tel: +886 2 2652 3052 E-mail: wtarn@ibms.sinica.edu.tw *These authors contributed equally to this work (Received 3 November 2008, revised 14 December 2008, accepted 9 January 2009) doi:10.1111/j.1742-4658.2009.06894.x Viruses take advantage of cellular machineries to facilitate their gene expression in the host. SR proteins, a superfamily of cellular precursor mRNA splicing factors, contain a domain consisting of repetitive argi- nine ⁄ serine dipeptides, termed the RS domain. The authentic RS domain or variants can also be found in some virus-encoded proteins. Viral pro- teins may act through their own RS domain or through interaction with cellular SR proteins to facilitate viral gene expression. Numerous lines of evidence indicate that cellular SR proteins are important for regulation of viral RNA splicing and participate in other steps of post-transcriptional viral gene expression control. Moreover, viral infection may alter the expression levels or modify the phosphorylation status of cellular SR proteins and thus perturb cellular precursor mRNA splicing. We review our current understanding of the interplay between virus and host in post-transcriptional regulation of gene expression via RS domain-containing proteins. Abbreviations CTE, constitutive transport element; E4, early region 4; EV, epidermodysplasia verruciformis; HBV, hepatitis B virus; HCV, hepatitis C virus; hnRNP, heterogeneous nuclear ribonucleoprotein; HPV, human papillomavirus; HSV, herpes simplex virus; IRES, internal ribosome entry site; N, nucleocapsid; PP, protein phosphatase; Background Autism spectrum disorders (ASD) is a collective term used to describe neurodevelopmental disorders with a pattern of qualitative abnormalities in three functional domains: reciprocal social interactions, communication, and restrictive interests and/or repetitive behaviors [1].  ere is strong evidence that 10 to 15% of ASD cases may be etiologically related to known genetic disorders, such as fragile X syndrome, tuberous sclerosis complex, and Rett syndrome [2,3]. However, the etiology of ASD in Abstract Background: Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by abnormalities in reciprocal social interactions and language development and/or usage, and by restricted interests and repetitive behaviors. Di erential gene expression of neurologically relevant genes in lymphoblastoid cell lines from monozygotic twins discordant in diagnosis or severity of autism suggested that epigenetic factors such as DNA methylation or microRNAs (miRNAs) may be involved in ASD. Methods: Global miRNA expression pro ling using lymphoblasts derived from these autistic twins and una ected sibling controls was therefore performed using high-throughput miRNA microarray analysis. Selected di erentially expressed miRNAs were con rmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis, and the putative target genes of two of the con rmed miRNA were validated by knockdown and overexpression of the respective miRNAs. Results: Di erentially expressed miRNAs were found to target genes highly involved in neurological functions and disorders in addition to genes involved in gastrointestinal diseases, circadian rhythm signaling, as well as steroid hormone metabolism and receptor signaling. Novel network analyses of the putative target genes that were inversely expressed relative to the relevant miRNA in these same samples further revealed an association with ASD and other co-morbid disorders, including muscle and gastrointestinal diseases, as well as with biological functions implicated in ASD, such as memory and synaptic plasticity. Putative gene targets (ID3 and PLK2) of two RT-PCR-con rmed brain- speci c miRNAs (hsa-miR-29b and hsa-miR-219-5p) were validated by miRNA overexpression or knockdown assays, respectively. Comparisons of these mRNA and miRNA expression levels between discordant twins and between case- control sib pairs show an inverse relationship, further suggesting that ID3 and PLK2 are in vivo targets of the respective miRNA. Interestingly, the up-regulation of miR-23a and down-regulation of miR-106b in this study re ected miRNA changes previously reported in post-mortem autistic cerebellum by Abu-Elneel et al. in 2008. This  nding validates these di erentially expressed miRNAs in neurological tissue from a di erent cohort as well as supports the use of the lymphoblasts as a surrogate to study miRNA expression in ASD. Conclusions: Findings from this study strongly suggest that dysregulation of miRNA expression contributes to the observed alterations in gene expression and, in turn, may lead to the pathophysiological conditions underlying autism. Investigation of post-transcriptional gene regulatory networks associated with autism spectrum disorders by microRNA expression pro ling of lymphoblastoid cell lines Tewarit Sarachana 1 , Rulun Zhou 2 , Guang Chen 2 , Husseini K Manji 2 and Valerie W Hu 1 * RESEARCH Open Access *Correspondence: bcmvwh@gwumc.edu 1 Department of Biochemistry and Molecular Biology, The George Washington University Medical Center, 2300 Eye St NW, Washington, DC 20037, USA Full list of author information is available at the end of the article © 2010 Sarachana et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction .. .Eukaryotic Post- transcriptional Gene Regulation regulation, with the frequency of different splicing alternatives controlled... evolve without eliminating the original, functional protein Link to Learning 2/5 Eukaryotic Post- transcriptional Gene Regulation Visualize how mRNA splicing happens by watching the process in action... proteins at the 5' or 3' UTR influences the stability of the RNA molecule 3/5 Eukaryotic Post- transcriptional Gene Regulation RNA Stability and microRNAs In addition to RBPs that bind to and control