Translation Termination

The pathway acts on PTCs originated from genes that are mutated or harboring errors occurring during pre-mRNA synthesis or maturation, and information technology acts during translation of an mRNA containing a PTC.

From: Progress in Brain Research , 2012

Fidelity and Quality Control in Gene Expression

Richard J. Jackson , ... Tatyana Five. Pestova , in Advances in Poly peptide Chemistry and Structural Biological science, 2012

Abstruse

Translation termination in eukaryotes occurs in response to a stop codon in the ribosomal A-site and requires two release factors (RFs), eRF1 and eRF3, which demark to the A-site as an eRF1/eRF3/GTP complex with eRF1 responsible for codon recognition. After GTP hydrolysis by eRF3, eRF1 triggers hydrolysis of the polypeptidyl-tRNA, releasing the completed poly peptide product. This leaves an 80S ribosome withal bound to the mRNA, with deacylated tRNA in its P-site and at least eRF1 in its A-site, which needs to be disassembled and released from the mRNA to permit further rounds of translation. The first step in recycling is dissociation of the 60S ribosomal subunit, leaving a 40S/deacylated tRNA complex bound to the mRNA. This is mediated past ABCE1, which is a somewhat unusual member of the ATP-binding cassette family of proteins with no membrane-spanning domain but 2 essential fe–sulfur clusters. Two distinct pathways accept been identified for subsequent ejection of the deacylated tRNA followed past dissociation of the 40S subunit from the mRNA, one executed by a subset of the canonical initiation factors (which therefore starts the process of preparing the 40S subunit for the side by side round of translation) and the other by Ligatin or homologous proteins. However, although this is the normal sequence of events, there are exceptions where the termination reaction is followed by reinitiation on the same mRNA (usually) at a site downstream of the stop codon. The overwhelming majority of such reinitiation events occur when the 5′-proximal open up reading frame (ORF) is short and can result in significant regulation of translation of the protein-coding ORF, but there are besides rare examples, mainly bicistronic viral RNAs, of reinitiation after a long ORF. Here, nosotros review our electric current agreement of the mechanisms of termination, ribosome recycling, and reinitiation afterward translation of short and long ORFs.

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Fidelity and Quality Control in Gene Expression

Marina 5. Rodnina , in Advances in Poly peptide Chemical science and Structural Biology, 2012

A Mechanism of Termination

Translation termination occurs when the ribosome encounters a stop codon (UAG, UAA, or UGA) in the A site. Finish codons in bacteria are recognized by RF1 and RF2: RF1 recognizes UAG and UAA codons, whereas RF2 recognizes UGA and UAA. Upon stop-codon recognition, RF1 and RF2 promote the hydrolysis of the ester bail in peptidyl–tRNA in the P site, leading to the release of the completed protein and the termination of protein synthesis. Afterwards, the dissociation of RF1 and RF2 from the ribosome is stimulated by RF3, a nonessential GTPase constitute in a subset of leaner ( Fig. vii; for review, meet Petry et al., 2008; Zaher and Dark-green, 2009a,b).

Fig. 7. Mechanism of stop-codon recognition by RF1 and RF2. Dissociation of the factors is facilitated past RF3 (non shown).

RF1 and RF2 are homologous proteins composed of 4 singled-out domains. Two sequence motifs are of detail functional importance: motifs PVT and SPF in domains two of RF1 and RF2, respectively, that are involved in the stop-codon recognition on the 30S subunit (Ito et al., 2000), and the GGQ motif in domain three that is disquisitional for catalysis of peptide release on the 50S subunit. The N-terminal domain 1 is required for RF association with RF3 just is dispensable for the main events of peptide release, while domain 4 is a structural domain packed confronting domain two. RF1 and RF2 can adopt ii distinct conformations, a closed conformation which is assumed in the absenteeism of the ribosome and incompatible with positioning the functional motifs in the advisable ribosomal sites (Vestergaard et al., 2001; Shin et al., 2004), and an extended conformation institute in the complexes of the factors with the ribosome (Petry et al., 2005; Korostelev et al., 2008, 2010; Laurberg et al., 2008; Weixlbaumer et al., 2008; Jin et al., 2010). The verbal timing of different termination steps (binding of RF1 or RF2 to the ribosome, the conformational change of the factor, and recognition of the cease codon) is not known and the exact role of RF3 uncertain (see Section IV.Grand).

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Molecular Prison cell Biology

W. Hu , in Encyclopedia of Cell Biology, 2016

Translation Termination and mRNA Stability

Eukaryotic mRNA translation termination requires 2 release factors, eRF1 and eRF3. Translation termination procedure can influence mRNA one-half-life. Specifically, information technology was observed that the N-concluding domain of eRF3, which is not required for translation termination, tin interact with Pab1, and this interaction is involved in modulating mRNA stability ( Hosoda et al., 2003). Disruption of this interaction results in translation-dependent stabilization of mRNA caused by decreased deadenylation rate (Hosoda et al., 2003). Interestingly, information technology was further found that sure deadenylase complexes can also demark to the same site on Pab1 that is involved in the interaction with eRF3 (Funakoshi et al., 2007). Thus, it has been postulated that eRF3 can regulate mRNA deadenylation by competitively binding to the Pab1, which and so modulates the recruitment and activation of deadenylase complexes (Funakoshi et al., 2007). In improver to the release factors, other proteins that tin can modulate translation termination tin can too influence mRNA stability. For example, a contempo characterized poly peptide named Tpa1 can interact with the 2 release factors and regulate the readthrough of stop codons (Keeling et al., 2006). Interestingly, although the detailed mechanisms yet remain elusive, knocking out this protein can take decreased deadenylation rate and increased mRNA stability (Keeling et al., 2006). Collectively, these results advise that mRNA translation termination can results in mRNP conformational changes that can influence mRNA stability, likely via modulating mRNA deadenylation.

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Translation Termination and Ribosome Recycling

Nadja Koloteva-Levin , Mick F. Tuite , in Encyclopedia of Biological Chemistry, 2004

Release Factors

Prokaryotic Polypeptide Chain Release Factors

In bacteria, translation termination is controlled by three different RFs ( Tabular array I). Two course-I protein release factors, RF1 and RF2, each decode ii of 3 finish codons, UAA or UAG (RF1) and UAA or UGA (RF2). Recognition of the stop codon by the RFs is mediated via a conserved tripeptide motif: Pro-Ala-Thr (PAT) in RF1 and Ser-Pro-Phe (SPF) in RF2. These tripeptides are referred to as peptide anticodons. The other important functional domain of class-I RFs contains the highly conserved amino acid motif Gly-Gly-Gln (GGQ) and it is this domain that triggers hydrolysis of the protein-tRNA bond. An understanding of how RFs trigger the release of the completed polypeptide from the tRNA at the P site has come up from a study of RF2 and its interaction with the terminating ribosome. When RF2 binds to the ribosome with a stop codon positioned at the A site, RF2 changes its iii-dimensional conformation such that the domain with the conserved "peptide anticodon" (SPF) interacts with the mRNA at the decoding heart, and the GGQ-containing domain comes in the contact with the ribosomal PTC to trigger hydrolysis of the peptidyl-tRNA linkage (Figure 2) .

Figure 2. The 3-dimensional conformation of a release factor changes once it is bound to the ribosome. (A) The RF2:bacterial ribosome complex indicating the points of contact between RF2 and the decoding center and the PTC of the ribosome. (B) The iii-dimensional structures of the unbound and leap forms of bacterial RF2 indicating the location of the "peptide anticodon" sequence Ser-Pro-Phe (SPF) and the conserved Gly-Gly-Gln (GGQ) amino acid motif.

The single class-II RF in bacteria, namely RF3, is a GTP-bounden protein that accelerates dissociation of either RF1 or RF2 from the ribosomal A-site after release of the completed polypeptide chain from the ribosome. RF3 bound to GDP accesses the ribosome which, in complex with RF1 or RF2, acts as guanine nucleotide substitution factors (GEFs) and triggers dissociation of GDP from RF3. This leads to the germination of a stable ribosome-RF1 (or RF2-) RF3 circuitous. Hydrolysis of the peptidyl-tRNA linkage triggered by RF1 or RF2 allows GTP binding to RF3. This induces an contradistinct RF3 conformation with a high affinity for the ribosome and leads to rapid dissociation of RF1 or RF2 from the termination complex. To leave the ribosome, RF3 requires GTP hydrolysis, which converts it to the Gross domestic product-bound course of RF3 which has a lower affinity for the ribosome. Once RF3 leaves the ribosome, it is ready to enter the next translation cycle.

Eukaryotic Chain Release Factors

In dissimilarity to bacteria, in eukaryotic cells translation is terminated past a single heterodimer consisting of two different RFs, eRF1 and eRF3. eRF1 is a class-I RF that decodes all three stop codons and triggers peptidyl-tRNA hydrolysis by the ribosome to release the nascent polypeptide. In eRF1, the finish codon recognition site is located close to the amino terminus of the poly peptide molecule in a region that contains an evolutionarily conserved tetrapeptide sequence, Asn-Ile-Lys-Ser (NIKS). This sequence may be functionally equivalent to the bacterial RF peptide anticodon. The GGQ motif institute in bacterial RF1 and RF2 is located in a separate domain of the protein to the NIKS sequence. The carboxy-terminal part of the eRF1 binds eRF3. The crystal structure of human being eRF1 shows that it is a Y-shaped molecule that resembles the structure of a tRNA (Effigy iii) . Since both bacterial and eukaryotic class-I RFs conduct out essentially the aforementioned office, i would expect them to interact similarly with ribosomal A site. As with RF2 (run across Figure 2), eRF1 must also undergo a conformational alter after binding to the ribosome in order that it can interact with both the ribosomal decoding site and the ribosomal PTC.

Effigy 3. Many of the protein factors involved in translation elongation and termination accept three-dimensional shapes that mimic a tRNA molecule. (A) Three-dimensional construction of a tRNA showing the location of the anticodon and the site to which the amino acid is covalently fastened. (B) The iii-dimensional structures of the mammalian RF eRF1 (left) and the bacterial translation elongation factor EF-Chiliad (right). The position of the conserved NIKS and GGQ amino acid motifs are indicated on eRF1, together with the domain to which the course-Two gene eRF3 binds.

The eukaryotic class-Two RF, eRF3, forms a complex with eRF1, and via this interaction enhances the efficiency of the translation termination although its function remains to exist fully defined. The GTPase activity of eRF3, which is triggered by terminate codons, is both eRF1- and ribosome dependent. The carboxy-terminal one-half of the eRF3 molecule shows significant amino acid identity to the translation elongation factor eEF1A that is responsible for bringing the aminoacyl tRNAs to the ribosome during polypeptide chain elongation. This suggests that eRF3 may act in a manner coordinating to eEF1A, a protein gene bringing the RF circuitous to the ribosome. A number of functions have been attributed to eRF3, e.g., it may displace eRF1 from the ribosome (i.e., the function assigned to prokaryotic RF3), or proofread the eRF1:terminate codon interaction, or stimulate ribosome disassembly (i.e., the office assigned to prokaryotic RRF; come across below), although directly experimental proof for any ane specific part is lacking. In mammalian cells, eRF3 binds to the poly(A)-binding protein (PABP), a protein associated with the poly(A) tails located at the 3′ end of the majority of eukaryotic mRNAs. This interaction might exist important for the regulation of both mRNA stability and translation initiation, since disruption of the eRF3::PABP interaction suppresses the recycling of the ribosome on the same mRNA.

In Bakers' yeast (Saccharomyces cerevisiae), the eRF3 poly peptide (besides known equally Sup35p) has an boosted remarkable belongings; information technology is a prion protein that acts every bit the protein determinant of a not-Mendelian genetic element called [PSI +]. Like the mammalian prion protein PrP, eRF3 can exist in one of two different conformations with the aggregation-decumbent prion conformer catalyzing the conversion of the normal soluble form, to the prion class. In [PSI +] cells the majority of eRF3 is found as an inactive, high molecular weight complex, and thus [PSI +] cells have a defect in translation termination albeit without detriment to yeast jail cell growth. This machinery may represent a novel means of regulating the efficiency of translation termination in yeast. There is no bear witness that mammalian eRF3 is a prion.

In Archaebacteria, the single course-I RF found (aRF1) shares significant amino acrid sequence and structural homology with eRF1, just non with either RF1 or RF2. Like eRF1, aRF1 is able to decode all three stop codons. No eRF3 homologue has been identified in any Archaebacterial genome.

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RNA helicases

Francesca Fiorini , ... Hervé Le Hir , in Methods in Enzymology, 2012

Abstruse

Degradation of eukaryotic mRNAs harboring a premature translation termination codon is ensured by the process of nonsense-mediated mRNA decay (NMD). The main effector of this quality-control pathway is the conserved RNA helicase UPF1 that forms a surveillance complex with the proteins UPF2 and UPF3. In all the organisms tested, the ATPase activity of UPF1 is essential for NMD. Here, we describe the expression of active recombinant UPF proteins and the reconstitution of the surveillance complex in vitro. To empathize how UPF1 is regulated during NMD, we developed unlike biochemical approaches. We describe methods to monitor UPF1 bounden to RNA, ATP hydrolysis and RNA unwinding in the presence of its binding partner UPF2. This functional analysis is an important complement for structural studies of protein complexes containing RNA helicases.

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Organellar and Metabolic Processes

William Zerges , Charles Hauser , in The Chlamydomonas Sourcebook, 2009

D. Termination

Since Chlamydomonas encodes close homologues of the eubacterial termination factors, translation termination in chloroplasts is likely to be mechanistically like (reviewed in Ramakrishnan, 2002). Translation termination begins when a finish codon is encountered in the A-site of the ribosome. Ii "class I" release factors, RF1 (which recognize UAA and UAG) and RF2 (which recognizes UAA) together with the "course 2" release gene RF3, release the completed polypeptide. Binding of RF1/2 to a ribosome triggers the hydrolysis and release of the peptide concatenation from the tRNA in the P site. RF3 promotes rapid dissociation of RF1 and RF2. Afterwards release of the peptide concatenation, the ribosome is left with mRNA and a deacylated tRNA in the P site. Ribosome recycling cistron (RRF) and EF-Yard are required to detach this circuitous (Table 28.iv; Rolland et al., 1999).

In Arabidopsis, screens of albino and loftier chlorophyll fluorescence (hcf) mutants uncovered genes for both RRF1 and RF2 (Meurer et al., 2002; Motohashi et al., 2007). RRF1 was uncovered through a screen of albino or pale-green (apg) mutants, where apg3-one carried a mutation in the chloroplast homologue AtcpRF1 (Motohashi et al., 2007). Complementation analysis using the E. coli rf1 mutant revealed that APG3 does function as an RF1 in East. coli. Since the chloroplasts of apg3-1 plants contained few internal thylakoid membranes, and chloroplast proteins related to photosynthesis were not detected by immunoblot analysis, AtcpRF1 is essential for chloroplast development.

The hcf109 mutation identified a chloroplast-targeted RF2-like protein (AtpfrB), whose absence causes decreased stability of UGA-containing mRNAs (Meurer et al., 2002). This implicates AtpfrB in the regulation of both mRNA stability and protein synthesis. Chlamydomonas may have lost this "auxiliary" function of RF2 as only two ORFs in the plastome encode UGA termination codons (Maul et al., 2002). If this is the case, reporter genes containing TGA stop codons would be predicted to non function because of a failure to terminate translation. There are two reports that accept shown that UGA stop codons tin can be functional in Chlamydomonas chloroplasts (Lee et al., 1996; Sizova et al., 1996). However, in both of these reports, the possibility of frameshifting and readthrough was non investigated.

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mRNA Turnover in Saccharomyces cerevisiae

Stuart W. Peltz , Allan Jacobson , in Command of Messenger RNA Stability, 1993

D trans-Acting Factors Involved in the Nonsense-Mediated mRNA Decay Pathway

Nonsense suppressors in yeast are either tRNA mutants, capable of decoding a translation termination codon, or mutants with lesions in not-tRNA genes, which enhance the expression of nonsense-containing alleles past other mechanisms. The latter mutants include the allosuppressors, frameshift suppressors, and omnipotent suppressors ( Surguchov, 1988; Hinnebusch and Liebman, 1991). At to the lowest degree one of these suppressors, upf1, acts by suppressing nonsense-mediated mRNA disuse.

Mutants in the UPF1 factor (and in UPF2, -3, and -4) were isolated on the footing of their ability to act as allosuppressors of the his4-38 frameshift mutation (Culbertson et al., 1980). The latter mutation is a single Thou insertion in the HIS4 factor that generates a   +   1 frameshift and a UAA nonsense codon in the triplet side by side to the insertion (Donahue et al., 1981). At 30°C, but not 37°C, his4-38 is suppressed past SUF1-1, which encodes a glycine tRNA capable of reading a 4 base of operations codon (Mendenhall et al., 1987). Mutations in UPF1 allow cells that are his4-38 and SUF1-one to grow at 37°C (Culbertson et al., 1980). The ground of this suppression appears to exist the loss of function of a trans-acting factor (Upf1p) essential for nonsense-mediated mRNA decay. Mutations in UPF1 lead to the selective stabilization of mRNAs containing early on nonsense mutations without affecting the decay rates of about other mRNAs (Table 2) (Leeds et al., 1991). Thus, in a UPF1 deletion mutant (upf1Δ), the his4-38 mRNA is stabilized approximately 5-fold, halflives of mRNAs from the PGK1 early nonsense alleles are stabilized approximately 12-fold, and half-lives of the PGK1 mRNAs with late nonsense codons or mRNAs from the wild-blazon MATαl, STE3, LEU2, HIS4, PGK1, PAB1, and ACT1 genes are unchanged (Table two). Regardless of position, all of the PGK1 nonsense alleles accept mRNA half-lives on the order of an hour in a upf1Δ strain (Peltz et al., 1993), a result that indicates that the loss of UPF1 office restores wild-type decay rates to mRNAs that would otherwise have been susceptible to the enhancement of decay rates promoted past early nonsense codons.

Suppression of nonsense-mediated mRNA decay in upf1Δ strains does not appear to result from enhanced read-through of the translation termination signal (Leeds et al., 1991), nor does it appear to be specific for a detail nonsense codon. The ability of upf1− mutants to suppress tyr7-1 (UAG), leu2-1 (UAA), leu2-2, (UGA), met8-1 (UAG), and his4-166 (UGA) (Leeds et al., 1992) indicates that they can act as omnipotent suppressors. Since many biosynthetic pathways exercise not require maximal levels of factor expression for cell survival (e.g., just half dozen% of the HIS4 gene production is required for growth on plates lacking histidine; Gaber and Culbertson, 1984), stabilization of nonsense-containing transcripts would allow synthesis of sufficient read-through protein to permit cells to grow on media lacking the relevant amino acids.

The UPF1 cistron has been cloned and sequenced and shown to be (one) nonessential for viability, (2) capable of encoding a 109-kDa poly peptide with both zinc finger and nucleotide (GTP) bounden site motifs, and (three) partially homologous to the yeast SEN1 gene (Leeds et al., 1992). The latter encodes a noncatalytic subunit of the tRNA splicing endonuclease complex (Winey and Culbertson, 1988), suggesting that Upf1p may too be role of a nuclease circuitous targeted to nonsense-containing mRNAs. It is unlikely, nevertheless, that its normal office is anticipatory, i.e., that it is solely involved in the degradation of mRNAs with premature nonsense codons. Ane part of the UPF1 gene may exist to regulate the decay rates of transcripts with upstream open reading frames, a determination that followed from an analysis of the steady-country levels and decay rates of mRNAs encoding gene products involved in the pyrimidine biosynthetic pathway (S. W. Peltz, A. H. Brown, J. Forest, A. Atkins, P. Leeds, G. Culbertson, and A. Jacobson, unpublished experiments). Transcription of several genes in this pathway (URA1, URA3, and URA4) is governed by the positive activator, PPR1 (Losson et al., 1983; Kammerer et al., 1984; Liljelund et al., 1984). The PPR1 mRNA itself has a pocket-sized (five codon) open up reading frame upstream of the PPR1 coding sequence; the termination codon for the upstream open reading frame overlaps with the ATG of the PPR1 coding sequence (Losson et al., 1983). Measurement of PPR1 mRNA disuse rates demonstrates that it decays threefold more slowly in a upf1− strain than in a UPF1+ strain [t 1/2  =   3   min (upf1− ) vs t 1/2  =   1   min (UPF1+ ); S. West. Peltz, A. H. Brown, J. Wood, A. Atkins, P. Leeds, Thousand. Culbertson, and A. Jacobson, unpublished experiments], suggesting that upstream open up reading frames, in addition to reducing the frequency of downstream translational initiation (Kozak, 1991; Hinnebusch and Liebman, 1991), may also destabilize specific transcripts. This destabilization machinery, which would involve the UPF1 gene product, would non necessarily pertain to all mRNAs with upstream open reading frames since at that place is no effect of UPF1 condition on the disuse rate of the GCN4 mRNA (Table 2; South. Due west. Peltz, A. H. Brown, J. Wood, A. Atkins, P. Leeds, M. Culbertson, and A. Jacobson, unpublished experiments).

The possibility as well exists that mRNAs with splicing errors constitute another class of UPF1 substrate; i.e., splicing fidelity would be maintained past the rapid turnover of RNAs in which processing errors take caused the insertion or deletion of ane or more nucleotides. At present, there is no evidence to suggest an error-decumbent splicing procedure. However, a related miracle, premature transport of pre-mRNA from the nucleus, may occur (Yost and Lindquist, 1988; reviewed in Kozak, 1991). Translation of intron-containing RNAs would virtually certainly lead to premature termination and, thus, could also be subject area to UPF1 regulation. Perhaps one general function of the UPF1 pathway is to ensure that aberrant proteins do not accumulate in those instances in which regulated splicing (Baker, 1989) or incomplete RNA editing (Simpson and Shaw, 1989; Stuart, 1991) generates mRNAs defective a functional open up reading frame.

Regardless of its physiological part, identification of the upf1 loss-of-part mutations provides a valuable tool to written report the nonsense-mediated mRNA decay pathway, cis-interim elements that promote nonsense-mediated mRNA decay can now exist defined as sequences that accelerate mRNA decay in wild-type cells, but that are inactivated in strains deleted for the UPF1 gene.

As noted higher up, iii other complementation groups of UPF mutants were identified in the same selection that identified upf1 (Culbertson et al., 1980). Since mutations in at least 1 of these genes, UPF3, also appear to reduce the rate of nonsense-mediated mRNA disuse (Leeds et al., 1992), it is likely that yeast genetics will evidence invaluable in the dissection of this mRNA degradation pathway.

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General considerations

Ivo F.A.C. Fokkema , Johan T. den Dunnen , in Clinical Dna Variant Interpretation, 2021

three′ untranslated region and the polyadenylation point

The 3′ untranslated region (3′ UTR ), the sequence after the translation termination (end) codon, contains several regulatory elements, such as bounden sites for miRNAs and RNA-bounden proteins, and the polyadenylation point and addition site. Together, these directly or indirectly influence RNA stability, folding, transport, localization, and translation efficiency and consequently RNA and protein levels [4]. Every bit the functional annotation of these elements (except for the polyadenylation signal) is largely defective, variants in this region are rarely considered as having deleterious consequences.

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Fidelity and Quality Control in Gene Expression

Brian D. Janssen , Christopher S. Hayes , in Advances in Protein Chemistry and Structural Biology, 2012

A Inefficient Translation Termination and A-site mRNA Cleavage

Leif Isaksson and colleagues first discovered that the concluding two residues of the nascent chain influence translation termination ( Mottagui-Tabar et al., 1994; Bjornsson et al., 1996). In general, C-last Pro, Asp, and Gly residues interfere with RF activeness and promote stop-codon readthrough. These nascent peptide sequences also induce ssrA-tagging of full-length proteins in E. coli, with C-concluding Asp-Pro and Pro-Pro sequences leading to specially high levels of tagging (Roche and Sauer, 2001; Hayes et al., 2002a). Ribosome-bound peptidyl prolyl-tRNA reacts with puromycin and aminoacyl-tRNA more slowly than other peptidyl-tRNA species (Muto and Ito, 2008; Wohlgemuth et al., 2008), perhaps accounting for the culling reactions that occur during termination in vivo. Nascent peptide-induced ribosome pausing is associated with cleavage of the A-site terminate-codon (Hayes and Sauer, 2003; Sunohara et al., 2004b). This RNase activity requires the ribosome and merely occurs in response to translational pausing (Hayes and Sauer, 2003; Janssen and Hayes, 2009). A-site cleavage products accumulate to high levels in ΔssrA cells, but are difficult to detect in ssrA + cells. Presumably, tmRNA activity facilitates the turnover of A-site truncated mRNA by releasing stalled ribosomes from the 3′-ends of these transcripts (Yamamoto et al., 2003). Together, these observations advise that A-site mRNA cleavage provides a mechanism for tmRNA recruitment when ribosomes pause on full-length transcripts. A-site mRNA cleavage activity is very similar to that of ribosome-dependent mRNA interferases like RelE and YoeB. However, A-site cleavage nevertheless occurs in cells lacking the RelE, MazF, ChpBK, YoeB, YafQ, and YhaV toxins (Hayes and Sauer, 2003; Garza-Sánchez et al., 2008). Because the known mRNA interferases are non involved, we and Hiroji Aiba'southward grouping proposed that the ribosome itself may catalyze this A-site cleavage reaction (Hayes and Sauer, 2003; Sunohara et al., 2004a).

Subsequent work has shown that A-site mRNA cleavage is a more complicated procedure than originally appreciated. A-site cleavage does not occur in Δrnb mutants, which lack RNase Two (Garza-Sánchez et al., 2009). RNase II is the major 3′-to-5′ exoribonuclease responsible for mRNA turnover in East. coli (Deutscher and Reuven, 1991). In Δrnb cells, prolonged translational pausing produces transcripts that are truncated +   12 nucleotides downstream of the A-site codon. This position corresponds to the ribosome leading border or "toeprint" (Yusupova et al., 2001), and suggests that downstream mRNA is degraded to the 3′-border of the stalled ribosome. The outcome of the Δrnb mutation on A-site mRNA cleavage is indirect because RNase 2 itself is unable to dethrone mRNA into the ribosomal A-site. Purified RNase 2 but degrades mRNA to the +   18 position with respect to the A-site codon inside the stalled ribosome (Garza-Sánchez et al., 2009). At that place are at least two models to explicate these findings. First, deletion of rnb could alter the expression of other RNases such that +   12 cleavage is favored over A-site mRNA cleavage. This model is consistent with microarray information showing that global transcription is significantly altered in E. coli Δrnb mutants (Mohanty and Kushner, 2003). Alternatively, RNase 2-mediated degradation of downstream mRNA could exist a precondition for further deposition into the A-site codon, mayhap by facilitating the activity of another RNase.

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High-Density Sequencing Applications in Microbial Molecular Genetics

Feng He , ... Allan Jacobson , in Methods in Enzymology, 2018

1 Introduction

Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance mechanism that targets mRNAs undergoing premature translation termination for rapid deposition ( He & Jacobson, 2015b; Kervestin & Jacobson, 2012; Lykke-Andersen & Bennett, 2014). The pathway was initially uncovered in Saccharomyces cerevisiae and Caenorhabditis elegans (Leeds, Peltz, Jacobson, & Culbertson, 1991; Peltz, Brown, & Jacobson, 1993; Pulak & Anderson, 1993) and later shown to exist conserved from yeast to humans (Behm-Ansmant et al., 2007; Schoenberg & Maquat, 2012). NMD'southward function was originally idea to be limited to quality control, i.e., the emptying of mRNAs derived from genes harboring nonsense mutations to prevent the accumulation of potentially deleterious truncated polypeptides (He, Peltz, Donahue, Rosbash, & Jacobson, 1993; Pulak & Anderson, 1993). Still, NMD also targets a significant fraction of apparently normal and physiologically functional wild-type mRNAs (Celik, Bakery, He, & Jacobson, 2017; Schweingruber, Rufener, Zund, Yamashita, & Muhlemann, 2013), indicating that it also serves as a fundamental posttranscriptional regulatory mechanism for eukaryotic gene expression.

In all organisms examined the activation of NMD requires a set of conserved core regulatory factors, Upf1, Upf2, and Upf3 (He & Jacobson, 2015b; Kervestin & Jacobson, 2012). These three proteins interact with each other, the ribosome, and multiple translation and mRNA disuse factors (Kervestin & Jacobson, 2012). Based on these molecular interactions, several potential functions have been proposed for the Upf factors, including remodeling terminating mRNPs (Franks, Singh, & Lykke-Andersen, 2010), releasing and recycling ribosomal subunits (Ghosh, Ganesan, Amrani, & Jacobson, 2010), and recruiting mRNA decay factors (He & Jacobson, 2015a; Nicholson, Josi, Kurosawa, Yamashita, & Muhlemann, 2014; Okada-Katsuhata et al., 2012). Notwithstanding, the exact roles for the Upfs, and their modes of action in NMD, remain largely unknown.

Transcripts targeted past NMD have been investigated using genome-wide high-density DNA microarrays over the concluding ii decades. Depending on the organism or prison cell type, NMD usually targets virtually five%–twenty% of the transcripts in a typical transcriptome (He et al., 2003; Lelivelt & Culbertson, 1999; Mendell, Sharifi, Meyers, Martinez-Murillo, & Dietz, 2004; Ramani et al., 2009; Rehwinkel, Letunic, Raes, Bork, & Izaurralde, 2005; Weischenfeldt et al., 2008) and these transcripts tin can be classified into several general categories. One category, exemplifying typical NMD substrates, includes mRNAs with a destabilizing premature termination codon (PTC) in their coding region. These transcripts are generated from endogenous genes harboring nonsense or frameshift mutations (He et al., 2003), pseudogenes (He et al., 2003; McGlincy & Smith, 2008), nonproductively rearranged genetic loci (Li & Wilkinson, 1998), or from alternative splicing events that lead to intron retention or inclusion of a PTC-containing exon (Jaillon et al., 2008; Lareau, Inada, Green, Wengrod, & Brenner, 2007; Lykke-Andersen et al., 2014; Ni et al., 2007). A 2nd category contains mRNA-similar transcripts with limited or no apparent coding potential, such every bit long noncoding RNAs (Kurihara et al., 2009; Lykke-Andersen et al., 2014; Tani, Torimura, & Akimitsu, 2013), minor RNAs derived from intragenic regions (Smith et al., 2014; Thompson & Parker, 2007), or transcripts of inactivated transposable elements (He et al., 2003). A third category contains a subset of physiologically relevant transcripts that appear to be "normal," such as mRNAs with upstream open reading frames (uORFs) (Arribere & Gilbert, 2013; Gaba, Jacobson, & Sachs, 2005; He et al., 2003), or with atypically long three′-UTRs (Kebaara & Atkin, 2009; Singh, Rebbapragada, & Lykke-Andersen, 2008), or normal-looking wild-type mRNAs with no singular features (He et al., 2003).

Loftier-density DNA microarrays have limited dynamic ranges and are likewise unable to distinguish different transcript isoforms originated from the same genetic locus or homologous loci. To generate a comprehensive and loftier-resolution itemize of NMD-regulated transcripts, and to delineate the defining features of these transcripts in NMD targeting, nosotros recently utilized RNA-Seq to reevaluate the effects of deleting the UPF1, UPF2, or UPF3 genes on the genome-broad expression of annotated yeast genes. Our new analyses confirm the previous results of microarray studies, but also uncover hundreds of new NMD-regulated transcripts that had escaped previous detection, including many intron-containing pre-mRNAs. Here, nosotros depict the detailed experimental methods and the bioinformatics pipeline of our RNA-Seq experiments for analyzing the endogenous NMD substrates in yeast cells. We present detailed procedures for RNA isolation and construction of RNA-Seq libraries from wild-blazon and upf1  Δ, upf2  Δ, or upf3  Δ cells, and as well outline the software used in our differential factor expression analyses.

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