What Happens to a Mrna Transcript Before It Can Be Translated?

RNA processing

Subrata Pal , in Fundamentals of Molecular Structural Biological science, 2020

Abstract

In eukaryotic cells, before RNA polymerase Two-generated transcripts could be translated into protein products, these transcripts (pre-mRNAs) need to be suitably processed to form messenger RNA (mRNA). Three major events constitute pre-mRNA processing: (a) five′-stop capping, (b) splicing, and (c) three′-end polyadenylation. In 5′-capping, the 5′-triphosphate of the nascent transcript is hydrolyzed to a diphosphate and a guanosine monophosphate is added in a opposite 5′-5′ orientation. Subsequently, the GpppN- cap is methylated to grade m⁷ GpppN-. In splicing, the noncoding sequences (introns) that dissever the coding sequences (exons) are removed and the exons are joined together. iii′-Polyadenylation involves cleavage of the transcript at a specific site and addition of a poly(A) tail. In addition, in that location is a fourth mechanism of RNA processing, known as RNA editing, in which instead of stretches of the transcript, private bases are inserted, deleted, or altered posttranscriptionally.

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Well-Known Combined Immune Deficiency Syndromes

John B. Ziegler , Sara Kashef , in Stiehm's Immune Deficiencies, 2014

Pathogenesis

RMRP is mainly located in the nucleolus in which it participates in generation of ribosomes past performing the endonucleolytic cleavage of the rRNA that ultimately leads to generation of mature 5.8S rRNA (Figure 6.4A). RMRP also processes mitochondrial RNA primers (an essential step to actuate mitochondrial DNA replication) and participates in cell wheel control by cleaving cyclin mRNA (Figure vi.4B). ¹⁰⁰ This is in accord with the generalized defect in cell growth observed in T cells, B cells, and fibroblasts, ^103–105 and could thus explain many of the features of CHH. Recently, an additional office was established by the ascertainment of the interaction of RMRP with the human being telomerase catalytic subunit (hTERT). ¹⁰⁶ Interestingly, the 3′ terminate of RMRP is essential for the germination and the activity of the hTERT–RMRP complex exhibiting RNA-dependent RNA polymerase activity. The hTERT–RMRP complex negatively regulates RMRP levels, simply may also accept an effect on other genes notwithstanding to be characterized. ¹⁰⁷

Effigy 6.four. Known function of the RNase MRP complex. (A) In human rRNA, cleavage at the upstream v.8S rRNA junction site is necessary for proper ribosome associates and is associated with the caste of bone dysplasia, (B) whereas mRNA cleavage of cyclin B2 mRNA is necessary for cell bike progression and is associated with the additional features like susceptibility to cancer, immune deficiency, anemia, and pilus hypoplasia. (C) At to the lowest degree the RMRP ortholog in yeast is involved in the processing of mitochondrial RNA, which functions equally primer for mitochondrial Dna replication. (D) Recently a new interaction with the human being telomerase catalytic subunit (hTERT) revealed an RNA-dependent RNA polymerase activity leading to siRNA altering gene expression.

Figure reproduced from Thiel and Rauch, ¹⁰⁷ with permission.

In CHH, in vitro lymphocyte proliferation is impaired and reduced secretion of IL-2 and IFN-γ and defective expression of IL-2 receptor α have been found. The proliferative defect cannot be rescued either by addition of exogenous IL-2 or by stimulation with phorbol myristate acetate and ionomycin – agents that featherbed receptor-mediated signaling. CHH patients accept a reduced number of naïve (CD45RA+) T lymphocytes. In that location appears to be increased apoptosis of circulating T lymphocytes associated with increased expression of Fas and Fas ligand (FasL) and of the proapoptotic molecule Bax, whereas expressions of the antiapoptotic molecules bcl-2 and inhibitory of apoptosis (IAP) are reduced. As mentioned above, RMRP allowed deficiency-causing mutations compromise cyclin B2 mRNA cleavage and may therefore result in increased levels of cyclin B2. Overexpression of this molecule leads to accumulation of cells in late mitosis and contributes to chromosomal instability. ¹⁰⁰

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Messenger RNA Processing in Eukaryotes

K. Potter , ... J.A. Wise , in Encyclopedia of Biological Chemistry (2d Edition), 2013

Abstruse

RNA processing is the term collectively used to describe the sequence of events through which the primary transcript from a cistron acquires its mature form. Very soon later synthesis by RNA polymerase Ii begins, transcripts from nuclear protein-coding genes larn a v′ cap construction. The 3′ finish of the messenger RNA (mRNA) is modified past the addition of a long string of adenosines in a process tightly linked to transcription termination. Finally, maturation of most eukaryotic mRNA precursors requires a process known equally splicing, in which internal noncoding segments known as introns are removed and the coding segments, known as exons, are joined to produce functional mRNAs. In complex eukaryotes, exons are much smaller than introns and provide the functional unit initially recognized past the splicing machinery. Because the splicing signals institute at the exon/intron boundaries have low information content, ancillary elements known every bit splicing enhancers and silencers are required to specify the precise sites of exon joining. Throughout their life cycles, mRNAs are busy with proteins, some stably bound and others associated only transiently; thus, the functional form recognized past both the RNA-processing machinery and the translating ribosome is a ribonucleoprotein circuitous (mRNP). An important advance in recent years is the discovery that RNA-processing events are mechanistically coupled to transcription by RNA polymerase II.

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Ribonucleases - Part B

Clare Simpson , David Stern , in Methods in Enzymology, 2001

A Role of RNA Processing and Decay in Regulating Chloroplast Gene Expression

RNA processing in chloroplasts includes mRNA 5′- and 3′-end processing, intron splicing, and intercistronic cleavages of polycistronic messages, every bit well equally typical tRNA and rRNA processing. These posttranscriptional steps, along with changes in RNA stability, take received considerable attention for 2 reasons. First, changes in chloroplast gene expression during chloroplast biogenesis or in response to environmental signals take much more often been shown to be posttranscriptional rather than transcriptional. Second, genetic studies designed to identify nuclear mutants defective in chloroplast function have nearly exclusively identified posttranscriptional defects. These information, along with in vitro systems developed to dissect the relevant mechanisms, have been the bailiwick of a number of reviews. ^ane−6

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Transcription | Messenger RNA Processing in Eukaryotes☆

Jo Ann Wise , Hua Lou , in Encyclopedia of Biological Chemical science (3rd Edition), 2021

Abstract

RNA processing is the term collectively used to describe the sequence of events through which the primary transcript from a cistron acquires its mature form. Very soon after synthesis past RNA polymerase Ii begins, pre-messenger RNAs transcribed from eukaryotic nuclear protein-coding genes larn a v׳ cap construction. Maturation in almost cases requires splicing, a process through which internal non-coding segments (introns) are removed and coding segments (exons) are joined to produce functional mRNAs for export to the cytoplasm. Formation of the iii′ terminate begins with endonucleolytic cleavage, which is tightly linked to transcription termination, followed past the improver of a poly(A) tail. In complex eukaryotes, multiple mRNAs tin be produced from a given gene transcript through a process known as alternative splicing. While the unlike steps in mRNA biogenesis were originally studied in isolation, it is now understood that there is coupling between transcription and processing, and that unlike processing events are as well coordinated. Throughout their lifetimes, eukaryotic transcripts associate dynamically with different sets of proteins, beginning with the co-transcriptional recognition of RNA processing signals via RNA-RNA or RNA-protein interactions. Whereas components of the cadre splicing and polyadenylation mechanism are deposited on all nascent transcripts, differential association of other RNA binding proteins tin can influence culling processing in the nucleus or translation in the cytoplasm. Each mRNA has a characteristic lifetime that ends with degradation past exonucleases.

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mRNA iii' Terminate Processing and Metabolism

Hari Krishna Yalamanchili , ... Zhandong Liu , in Methods in Enzymology, 2021

iv.7.i Integration with CLIP-seq data

RNA processing readouts like alternative splicing and alternative polyadenylation can exist substantiated by matching Clip-seq ( Stork & Zheng, 2016) bespeak near the splice sites or cleavage sites (Zhu et al., 2018), respectively. For demonstration purpose here we used publicly available NUDT21 CLIP-seq data in GEO database (GSM917661). Processing of Clip-seq information is described elsewhere (Uhl, Houwaart, Corrado, Wright, & Backofen, 2017). Fig. 6A and B illustrate three′UTR shrinking in PAK1 and VMA21, respectively. Corresponding NUDT21 bounden (Prune-seq indicate) in the proximity of the polyadenylation sites are marked with arrow heads. CLIP-seq signal substantiate the 3′UTR shrinking of PAK1 and VMA21 as the straight issue of NUDT21 KD.

Fig. vi. Integration with Clip-seq data: Prune point overlapping with PAC-seq signal for (A) PAK1 and (B) VMA21.

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Genomics in Multiple Myeloma

Francesca Cottini , ... Giovanni Tonon , in Cancer Genomics, 2014

Mutations in RNA Processing Genes

RNA processing is a make new field in cancer biology, starting with the identification of aberrancies in microRNA product and, more recently, highlighted past the discovery of mutations involving multiple components of the RNA splicing machinery such as U2AF35, ZRSR2, SRSF2 and SF3B1 in myelodysplastic syndromes and other cancers [73].

DIS3 is mutated in 10–18% of MM [65,74]. It encodes a highly conserved RNA exonuclease, which serves as the catalytic component of the exosome complex involved in regulation of processing and abundance of different RNA species. The four observed mutations occur at highly conserved regions and cluster within the RNA domain facing the enzyme's catalytic pocket. Thus, DIS3 mutations might deregulate protein translation and mRNA processing. Even though the precise function of DIS3 in MM pathogenesis is nonetheless unknown, patients whose MM bears DIS3 mutations often exhibit deletion of the remaining DIS3 allele, suggesting that these mutations tin can complete the loss of function of the protein. However, other evidence suggests that one of the mutations can correspond a gain of function. Another interesting finding is that DIS3 mutations are associated with deletions of the RB1 region, equally reported recently [74]. Whether these events are collaborative and may elicit a synergic effect in MM growth and progression is all the same unknown. In addition, mutation of DIS3 may exist enriched in MM with either a t(four;14) or t(11;xiv) [74].

FAM46C gene is mutated in 13% of patients, and its genomic locus is deleted in ten–15% of cases. FAM46C deletion is associated with a worse prognosis [46]. It belongs to the Ntase fold protein superfamily, which transfers NMP from NTP to an acceptor hydroxyl group of protein or nucleic acid. FAM46C function is still unknown, simply GSEA algorithms define a concomitant expression of FMA46C with a prepare of ribosomal proteins known to exist tightly co-regulated and involved in initiation and elongation of poly peptide translation. Similarly, FAM46C can besides comport every bit an mRNA stability factor [46,65].

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Ribonucleases - Part B

Karsten Liere , ... Gerhard Link , in Methods in Enzymology, 2001

In Vitro Redox Assays of p54

RNA-binding and processing assays are carried out in standard reaction mixtures containing p54 (see above). They are pretreated, however, with a 20   gM concentration of oxidant menadione (K3; Sigma), cystine (CysCys; Sigma), or oxidized glutathione (GSSG; Sigma), or with reductant two-mercaptoethanol (EtSH; Sigma), dithiothreitol (DTT; Sigma), cysteine (CysSH; Sigma), or reduced glutathione (GSH; Sigma). The pretreatments are at room temperature for 10   min before the add-on of labeled RNA. Stock solutions (100   mOne thousand) of the redox reagents are prepared in 50   yardG Tris-HCl, pH   8.0. Escherichia coli thioredoxin (five μM; BRL, Gaithersburg, Medico) is reduced with a 5000 Chiliad excess of DTT or is oxidized with a 5000-fold excess of menadione before apply.

To confirm that p54 processing activity is specifically modulated past glutathione, redox reversibility assays are carried out. Purified p54 is preincubated with 20   kM oxidized or reduced glutathione, menadione, or DTT for 5   min and is later treated with equimolar amounts of the indicated redox reagent for an additional 5   min (Fig. 2B).

To test the extent to which phosphorylation and redox land act together in the control of p54 activity in vitro, processing experiments are carried out with p54 that has been pretreated in various combinations (Fig. 2C). In experiments involving initial treatment with the kinase or CIAP, followed past the redox-reactive reagent, the two steps are separated past reisolation of the protein equally described above. In contrast, when p54 is starting time treated by redox reagents and and then subjected to phosphorylation or dephosphorylation, the latter treatments can be carried out without prior reisolation of p54. In the case of phosphorylation, after preincubation with the respective redox reagent, a mixture is added containing two μl of 10   ×   kinase buffer, 0.2 μfifty of 20   nThou ATP, and 0.five μg of PKA (20-μl final reaction volume; containing 10   ×   kinase buffer, 0.ii   thousandK ATP) and the sample is incubated at 30° for 30   min. Similarly, dephosphorylation later on redox handling is done by adding a mixture of 2 μl of 10   ×   CIAP buffer and 20   units of CIAP in a terminal reaction volume of 20 μl, and incubation of the sample is continued at 30° for 30   min. To start the subsequent RNA-binding or processing reactions, radioactively labeled RNA is added and the samples are incubated at room temperature according to the protocols given above.

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Rhabdovirus

Wang-Shick Ryu , in Molecular Virology of Man Pathogenic Viruses, 2017

14.iv Furnishings on Host

CPE: VSV-infected cells are lysed and as a consequence, plaques are formed. How does VSV infection lead to prison cell lysis? VSV infection blocks host jail cell protein synthesis, while viral protein synthesis remains unaffected. Two mechanisms for "host shutoff" office have been revealed, as shown beneath.

RNA Processing : First, VSV infection blocks the nuclear export of cellular pre-mRNA. Specifically, information technology was shown that viral M poly peptide binds to Rae1, ⁹ a nuclear export factor, thereby blocking the nuclear consign of cellular pre-mRNA (Fig. 14.seven). Information technology should be noted that this inhibitory mechanism does non touch on the viral mRNA synthesis, as the viral mRNA synthesis is limited to the cytoplasm. Hence, this machinery is also dubbed "host shutoff."

Figure 14.vii. Host shutoff functions by VSV Grand protein.

Cellular pre-mRNAs are exported to cytoplasm via nuclear pore. TAP/p15 complex serves as a transport receptor for pre-mRNAs, and recruits pre-mRNAs to NPC (nuclear pore complex). VSV M protein blocks the nuclear export of pre-mRNAs via its interaction with Rae1 molecule, a nuclear consign factor. Note that Rae1 acts equally a nuclear export factor via its interaction with Nup98, a component of NPC. In addition, VSV inhibits cellular translation via two mechanisms: (1) by enhancing eIF2 phosphorylation and (ii) by suppressing eIF4E phosphorylation.

Translation: In addition, VSV infection blocks cellular translation. First, the phosphorylation of eIF4E ¹⁰ is reduced in VSV-infected cells, thereby reducing eIF4F ¹¹ complex formation. On the other hand, the phosphorylation of eIF2 is increased in VSV-infected cells, thereby decreasing translation. Perhaps, the phosphorylation of eIF2 ¹² by IFN-activated PKR profoundly contributes to the translation suppression (see Fig. 5.8). An intriguing point is that VSV protein translation remains unaffected and the viral proteins accumulated, although host translation function is substantially impaired. The question of how selectively VSV mRNA translation is unaffected remains unknown.

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Epigenetic Shaping of Sociosexual Interactions

Ryohei Sekido , in Advances in Genetics, 2014

4.3 SRY May Regulate Pre-mRNA Splicing

RNA processing is besides of import to elicit different biological function from an RNA transcript. Several lines of testify demonstrate that RNA processing is crucial for sex decision. For example, in Drosophila, a ratio of Ten chromosomes to autosomes affects the expression of sex-lethal (Sxl) cistron that encodes an RNA-binding poly peptide. In females, a high level of SXL poly peptide promotes an alternative splicing of transformer (tra) pre-mRNA to exist translated into the functional TRA^F poly peptide, which is also an RNA-binding protein. TRA^F in turn promotes some other alternative splicing event to produce doublesex (dsx) mRNA for the female-specific grade of DSX protein (DSX^F) and fruitless mRNA that encodes no protein, while the dsx and fru genes produce the male-specific mRNAs for DSX^{One thousand} and FRU^Thou proteins in the absenteeism of TRA^F, respectively. The production of the correct course of DSX and the presence or absence of FRU in each sex controls sexual differentiation and sexual behavior. DSX controls sexual differentiation, including pheromone product, besides as sexual beliefs (Rideout, Dornan, Neville, Eadie, & Goodwin, 2010). FRU is a chromatin-associated protein required for the development of male courting circuits (Demir & Dickson, 2005; Ito et al., 2012; Kimura, Ote, Tazawa, & Yamamoto, 2005; Manoli et al., 2005).

In some cell lines, SRY protein colocalizes with β-catenin, splicing factor U2AF65, and RPS7 and RPL13a in nuclear speckles (Bernard et al., 2008; Ohe et al., 2002; Sato et al., 2011). It is known that ribosomal proteins are imported into the nucleus and assembled with ribosomal RNA to form spliceosomes. These suggest that SRY plays a role in culling pre-mRNA splicing, and indeed SRY facilitates alternative splicing of several well-known pre-mRNA substrates in an in vitro assay (Ohe et al., 2002). Information technology is unknown how SRY localizes in nuclear speckles. The prove that β-catenin changes its subcellular localization by posttranscriptional modifications (Brembeck et al., 2004) and physically interacts with SRY (Bernard et al., 2008), suggests that β-catenin recruits SRY to nuclear speckles. Moreover, an X-linked orphan nuclear receptor, Dax1, also acts as a shuttling RNA-binding protein between the nucleus and the cytoplasm (Lalli, Ohe, Hindelang, & Sassone-Corsi, 2000). Dax1 has been idea to be antagonistic to Sry activity in sex activity conclusion because its overexpression causes XY sex reversal in the presence of Sry (Swain, Narvaez, Burgoyne, Camerino, & Lovell-Badge, 1998), although the molecular ground of this animosity remains to exist explored. Recent studies have demonstrated that Dax1 also interacts with U2AF65 (Ohe, Tamai, Parvinen, & Sassone-Corsi, 2009), suggesting that Dax1 might inhibit SRY-mediated pre-mRNA splicing past competing with U2AF65.

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