Termination Readthrough Research Articles

The nucleosome DNA entry-exit site is important for transcription termination and prevention of pervasive transcription.

Compared to other stages in the RNA polymerase II transcription cycle, the role of chromatin in transcription termination is poorly understood. We performed a genetic screen in Saccharomyces cerevisiae to identify histone mutants that exhibit transcriptional readthrough of terminators. Amino acid substitutions identified by the screen map to the nucleosome DNA entry-exit site. The strongest H3 mutants revealed widespread genomic changes, including increased sense-strand transcription upstream and downstream of genes, increased antisense transcription overlapping gene bodies, and reduced nucleosome occupancy particularly at the 3' ends of genes. Replacement of the native sequence downstream of a gene with a sequence that increases nucleosome occupancy in vivo reduced readthrough transcription and suppressed the effect of a DNA entry-exit site substitution. Our results suggest that nucleosomes can facilitate termination by serving as a barrier to transcription and highlight the importance of the DNA entry-exit site in broadly maintaining the integrity of the transcriptome.

Intron retention is among six unreported AGL mutations identified in Malaysian GSD III patients.

Glycogen storage disease type III is an autosomal recessive disorder that is caused by deficiencies of the glycogen debranching enzyme. Mutations within the AGL gene have been found to be heterogeneous, with some common mutations being reported in certain populations. The mutation spectrum of AGL gene in the multi-ethnic Malaysian population is still unknown. The present study seeks to determine the mutation spectrum of the AGL gene in Malaysian population. A total of eleven patients (eight Malay, two Chinese and one Bajau) were investigated. Genomic DNA was extracted and subsequently the AGL gene was amplified using specific primers and sequenced. Mutations found were screened in 150 healthy control samples either by restriction enzyme digestion assay or TaqMan® SNP Genotyping assay. We identified six unreported mutations (c.1423+1G>T, c.2914_2915delAA, c.3814_3815delAG, c.4333T>G, c.4490G>A, c.4531_4534delTGTC) along with three previously reported mutations (c.99C>T, c.1783C>T, c.2681+1G>A). One of the six unreported mutation causes abnormal splicing and results in retention of intron 12 of the mature transcript, while another is a termination read-through. One of the reported mutation c.2681+1G>A was recurrently found in the Malay patients (n = 7 alleles; 31.8%). The mutation spectrum of the AGL gene in Malaysian patients has shown considerable heterogeneity, and all unreported mutations were absent in all 150 healthy control samples tested.

Regulation of the Elongation Phase of Protein Synthesis Enhances Translation Accuracy and Modulates Lifespan

Maintaining accuracy during protein synthesis is crucial to avoid producing misfolded and/or non-functional proteins. The target of rapamycin complex 1 (TORC1) pathway and the activity of the protein synthesis machinery are known to negatively regulate lifespan in many organisms, although the precise mechanisms involved remain unclear. Mammalian TORC1 signaling accelerates the elongation stage of protein synthesis by inactivating eukaryotic elongation factor 2 kinase (eEF2K), which, when active, phosphorylates and inhibits eEF2, which mediates the movement of ribosomes along mRNAs, thereby slowing down the rate of elongation. We show that eEF2K enhances the accuracy of protein synthesis under a range of conditions and in several cell types. For example, our data reveal it links mammalian (m)TORC1 signaling to the accuracy of translation. Activation of eEF2K decreases misreading or termination readthrough errors during elongation, whereas knocking down or knocking out eEF2K increases their frequency. eEF2K also promotes the correct recognition of start codons in mRNAs. Reduced translational fidelity is known to correlate with shorter lifespan. Consistent with this, deletion of the eEF2K ortholog or other factors implicated intranslation fidelity in Caenorhabditis elegans decreases lifespan, and eEF2K is required for lifespan extension induced by nutrient restriction. Our data uncover a novel mechanism linking nutrient supply, mTORC1 signaling, and the elongation stage of protein synthesis, which enhances the accuracy of protein synthesis. Our data also indicate that modulating translation elongation and its fidelity affects lifespan.

Eukaryotic translational termination efficiency is influenced by the 3' nucleotides within the ribosomal mRNA channel.

When a stop codon is at the 80S ribosomal A site, there are six nucleotides (+4 to +9) downstream that are inferred to be occupying the mRNA channel. We examined the influence of these downstream nucleotides on translation termination success or failure in mammalian cells at the three stop codons. The expected hierarchy in the intrinsic fidelity of the stop codons (UAA>UAG>>UGA) was observed, with highly influential effects on termination readthrough mediated by nucleotides at position +4 and position +8. A more complex influence was observed from the nucleotides at positions +5 and +6. The weakest termination contexts were most affected by increases or decreases in the concentration of the decoding release factor (eRF1), indicating that eRF1 binding to these signals was rate-limiting. When termination efficiency was significantly reduced by cognate suppressor tRNAs, the observed influence of downstream nucleotides was maintained. There was a positive correlation between experimentally measured signal strength and frequency of the signal in eukaryotic genomes, particularly in Saccharomyces cerevisiae and Drosophila melanogaster. We propose that termination efficiency is not only influenced by interrogation of the stop signal directly by the release factor, but also by downstream ribosomal interactions with the mRNA nucleotides in the entry channel.

The Role of +4U as an Extended Translation Termination Signal in Bacteria.

Termination efficiency of stop codons depends on the first 3′ flanking (+4) base in bacteria and eukaryotes. In both Escherichia coli and Saccharomyces cerevisiae, termination read-through is reduced in the presence of +4U; however, the molecular mechanism underlying +4U function is poorly understood. Here, we perform comparative genomics analysis on 25 bacterial species (covering Actinobacteria, Bacteriodetes, Cyanobacteria, Deinococcus-Thermus, Firmicutes, Proteobacteria, and Spirochaetae) with bioinformatics approaches to examine the influence of +4U in bacterial translation termination by contrasting highly- and lowly-expressed genes (HEGs and LEGs, respectively). We estimated gene expression using the recently formulated Index of Translation Elongation, ITE, and identified stop codon near-cognate transfer RNAs (tRNAs) from well-annotated genomes. We show that +4U was consistently overrepresented in UAA-ending HEGs relative to LEGs. The result is consistent with the interpretation that +4U enhances termination mainly for UAA. Usage of +4U decreases in GC-rich species where most stop codons are UGA and UAG, with few UAA-ending genes, which is expected if UAA usage in HEGs drives up +4U usage. In HEGs, +4U usage increases significantly with abundance of UAA nc_tRNAs (near-cognate tRNAs that decode codons differing from UAA by a single nucleotide), particularly those with a mismatch at the first stop codon site. UAA is always the preferred stop codon in HEGs, and our results suggest that UAAU is the most efficient translation termination signal in bacteria.

Ribosome profiling reveals an adaptation strategy of reduced bacterium to acute stress

Bacteria of class Mollicutes (mycoplasmas) feature significant genome reduction which makes them good model organisms for systems biology studies. Previously we demonstrated, that drastic transcriptional response of mycoplasmas to stress results in a very limited response on the level of protein. In this study we used heat stress model of M. gallisepticum and ribosome profiling to elucidate the process of genetic information transfer under stress. We found that under heat stress ribosomes demonstrate selectivity towards mRNA binding. We identified that heat stress response may be divided into two groups on the basis of absolute transcript abundance and fold-change in the translatome. One represents a noise-like response and another is likely an adaptive one. The latter include ClpB chaperone, cell division cluster, homologs of immunoblocking proteins and short ORFs with unknown function. We found that previously identified read-through of terminators contributes to the upregulation of transcripts in the translatome as well. In addition we identified that ribosomes of M. gallisepticum undergo reorganization under the heat stress. The most notable event is decrease of the amount of associated HU protein. In conclusion, only changes of few adaptive transcripts significantly impact translatome, while widespread noise-like transcription plays insignificant role in translation during stress.

NusA-dependent transcription termination prevents misregulation of global gene expression.

Intrinsic transcription terminators consist of an RNA hairpin followed by a U-rich tract, and these signals can trigger termination without the involvement of additional factors. Although NusA is known to stimulate intrinsic termination in vitro, the in vivo targets and global impact of NusA are not known because it is essential for viability. Using genome-wide 3' end-mapping on an engineered Bacillus subtilis NusA depletion strain, we show that weak suboptimal terminators are the principle NusA substrates. Moreover, a subclass of weak non-canonical terminators was identified that completely depend on NusA for effective termination. NusA-dependent terminators tend to have weak hairpins and/or distal U-tract interruptions, supporting a model in which NusA is directly involved in the termination mechanism. Depletion of NusA altered global gene expression directly and indirectly via readthrough of suboptimal terminators. Readthrough of NusA-dependent terminators caused misregulation of genes involved in essential cellular functions, especially DNA replication and metabolism. We further show that nusA is autoregulated by a transcription attenuation mechanism that does not rely on antiterminator structures. Instead, NusA-stimulated termination in its 5' UTR dictates the extent of transcription into the operon, thereby ensuring tight control of cellular NusA levels.

Loss of the Yeast SR Protein Npl3 Alters Gene Expression Due to Transcription Readthrough.

Yeast Npl3 is a highly abundant, nuclear-cytoplasmic shuttling, RNA-binding protein, related to metazoan SR proteins. Reported functions of Npl3 include transcription elongation, splicing and RNA 3’ end processing. We used UV crosslinking and analysis of cDNA (CRAC) to map precise RNA binding sites, and strand-specific tiling arrays to look at the effects of loss of Npl3 on all transcripts across the genome. We found that Npl3 binds diverse RNA species, both coding and non-coding, at sites indicative of roles in both early pre-mRNA processing and 3’ end formation. Tiling arrays and RNAPII mapping data revealed 3’ extended RNAPII-transcribed RNAs in the absence of Npl3, suggesting that defects in pre-mRNA packaging events result in termination readthrough. Transcription readthrough was widespread and frequently resulted in down-regulation of neighboring genes. We conclude that the absence of Npl3 results in widespread 3' extension of transcripts with pervasive effects on gene expression.

351. Clonal Analysis Allows Characterization and Risk Assessment of Viral-Cellular Fusion Transcripts Generated by Transcriptional Termination Read-Through in Keratinocytes Transduced with Self-Inactivating Retroviral Vectors

351. Clonal Analysis Allows Characterization and Risk Assessment of Viral-Cellular Fusion Transcripts Generated by Transcriptional Termination Read-Through in Keratinocytes Transduced with Self-Inactivating Retroviral Vectors

Revealing frequent alternative polyadenylation and widespread low-level transcription read-through of novel plant transcription terminators

Plant genetic engineering can create transgenic crops with improved characteristics by introducing trait genes through transformation. Appropriate regulatory elements such as promoters and terminators have to be present in certain configurations for the transgenes to be properly expressed. Five terminators native to soybean genes-encoding a MYB family transcription factor (MYB2), a Kunitz trypsin inhibitor (KTI1), a plasma membrane intrinsic protein (PIP1), a translation elongation factor (EF1A2) and a metallothionein protein (MTH1) were cloned and tested for their ability to enable transgene expression, mRNA polyadenylation and transcription termination. The terminators are as good as a control terminator of the potato proteinase inhibitor II gene (PINII) in conferring proper transgene expression, leading to mRNAs with various polyadenylation sites and terminating mRNA transcripts. RNA transcription read-through was detected in all transgenic plants and was quantified by qRT-PCR to be <1% at positions approximately 1 kb downstream of the 5' ends of different terminators. The detection of read-through RNA transcripts of the corresponding endogenous genes up to approximately 1 kb beyond the polyadenylation sites suggests that limited RNA transcription read-through is a normal phenomenon of gene expression. The study also provided more choices of terminators for plant genetic engineering when constructing DNA constructs containing multiple gene expression cassettes.

Transcriptional Analysis of the Genetic Element pSSVx: Differential and Temporal Regulation of Gene Expression Reveals Correlation between Transcription and Replication

pSSVx from Sulfolobus islandicus strain REY15/4 is a hybrid between a plasmid and a fusellovirus. A systematic study performed by a combination of Northern blot analysis, primer extension, and reverse transcriptase PCR revealed the presence of nine major transcripts whose expression was differentially and temporally regulated over the growth cycle of S. islandicus. The map positions of the RNAs as well as the clockwise and the anticlockwise directions of their transcription were determined. Some genes were clustered and appeared to be transcribed as polycistronic messengers, among which one long transcriptional unit comprised the genes for the plasmid copy number control protein ORF60 (CopG), ORF91, and the replication protein ORF892 (RepA). We propose that a termination readthrough mechanism might be responsible for the formation of more than one RNA species from a single 5' end and therefore that the nine different RNAs corresponded to only seven different transcriptional starts. Three transcripts, ORF76 and two antisense RNAs, countertranscribed RNA1 (ctRNA1) and ctRNA2, were found to be specifically expressed during (and hence correlated to) the phase in which the pSSVx copy number is kept under stringent control, as they were completely switched off upon the onset of the induction of replication.

Microarray detection of novel nuclear RNA substrates for the exosome

Microarray analyses were performed on yeast strains mutant for the nuclear-specific exosome components Rrp6p and Rrp47p/Lrp1p or the core component Rrp41p/Ski6p, at permissive temperature and following transfer to 37 degrees C. 339 mRNAs showed clearly altered expression levels, with an unexpectedly high degree of heterogeneity in the different exosome mutants. In contrast, no clear alterations were seen in strains lacking the cytoplasmic exosome component Ski7p. 27 mRNAs that were overexpressed in each strain defective in the nuclear exosome are good candidates for regulation by nuclear turnover. These included the mRNA for the autoregulated RNA-binding protein Nrd1p. Northern and primer extension analyses confirmed the elevated NRD1 mRNA levels in exosome mutants, and revealed the accumulation of truncated 5' fragments of the mRNA. These contain a predicted Nrd1p-binding site, potentially sequestering the protein and disrupting its autoregulation. Several genes located immediately downstream of independently transcribed snoRNA genes were overexpressed in exosome mutants, presumably due to stabilization of the products of transcription termination read-through. Further analyses indicated that many snoRNA and snRNA genes are inefficiently terminated, but read-through transcripts into downstream ORFs are normally rapidly degraded by the exosome.

Lack of Pseudouridine 38/39 in the Anticodon Arm of Yeast Cytoplasmic tRNA Decreases in Vivo Recoding Efficiency

Many different modified nucleotides are found in naturally occurring tRNA, especially in the anticodon region. Their importance for the efficiency of the translational process begins to be well documented. Here we have analyzed the in vivo effect of deleting genes coding for yeast tRNA-modifying enzymes, namely Pus1p, Pus3p, Pus4p, or Trm4p, on termination readthrough and +1 frameshift events. To this end, we have transformed each of the yeast deletion strains with a lacZ-luc dual-reporter vector harboring selected programmed recoding sites. We have found that only deletion of the PUS3 gene, encoding the enzyme that introduces pseudouridines at position 38 or 39 in tRNA, has an effect on the efficiency of the translation process. In this mutant, we have observed a reduced readthrough efficiency of each stop codon by natural nonsense suppressor tRNAs. This effect is solely due to the absence of pseudouridine 38 or 39 in tRNA because the inactive mutant protein Pus3[D151A]p did not restore the level of natural readthrough. Our results also show that absence of pseudouridine 39 in the slippery tRNA(UAG)(Leu) reduces +1 frameshift efficiency. Therefore, the presence of pseudouridine 38 or 39 in the tRNA anticodon arm enhances misreading of certain codons by natural nonsense tRNAs as well as promotes frameshifting on slippery sequences in yeast.

Gene overexpression as a tool for identifying new trans-acting factors involved in translation termination in Saccharomyces cerevisiae.

In eukaryotes, translation termination is dependent on the availability of both release factors, eRF1 and eRF3; however, the precise mechanisms involved remain poorly understood. In particular, the fact that the phenotype of release factor mutants is pleiotropic could imply that other factors and interactions are involved in translation termination. To identify unknown elements involved in this process, we performed a genetic screen using a reporter strain in which a leaky stop codon is inserted in the lacZ reporter gene, attempting to isolate factors modifying termination efficiency when overexpressed. Twelve suppressors and 11 antisuppressors, increasing or decreasing termination readthrough, respectively, were identified and analyzed for three secondary phenotypes often associated with translation mutations: thermosensitivity, G418 sensitivity, and sensitivity to osmotic pressure. Interestingly, among these candidates, we identified two genes, SSO1 and STU2, involved in protein transport and spindle pole body formation, respectively, suggesting puzzling connections with the translation termination process.

A novel stop codon readthrough mechanism produces functional Headcase protein in Drosophila trachea.

Translational regulation provides an efficient means to control the localization and production of proteins. The headcase (hdc) mRNA in Drosophila generates two overlapping proteins as a result of translational readthrough of an internal UAA stop codon. This readthrough event is necessary for the function of hdc as a branching inhibitor during tracheal development. By ectopic expression of different Hdc proteins in the trachea, we show that the long Hdc form alone, can function as a potent branching inhibitor whose activity is proportional to its amount. The suppression of termination in the hdc mRNA is not stop-codon dependent, suggesting that the readthrough does not involve codon specific suppressors. We have identified an 80 nucleotide sequence immediately downstream of the UAA, which is necessary and sufficient to confer termination readthrough in a heterologous mRNA. We present a novel mechanism of eukaryotic translational termination suppression that may regulate the amount of functional Hdc.

Modification of the Properties of Elongating RNA Polymerase by Persistent Association with Nascent Antiterminator RNA

Nascent RNA encoded by putL, a cis-acting antitermination site of bacteriophage HK022, increases readthrough of terminators by directly modifying the transcript elongation complex. To characterize the interaction between the antiterminator RNA and RNA polymerase, we stalled the elongation complex downstream of putL and determined the sensitivity of the transcript to ribonuclease cleavage. Part of PutL RNA was protected from cleavage by wild-type polymerase, but not by a mutant with a defect in put-dependent antitermination. We also exposed the stalled complex to oligonucleotides complementary to putL RNA, restarted transcription, and measured antitermination. Some, but not all, complementary oligonucleotides inhibited antitermination. Finally, cleavage of the RNA between putL and the 3′-end released putL RNA from the stalled complex and prevented antitermination.

Nonsense-mediated decay mutants do not affect programmed -1 frameshifting.

Sequences in certain mRNAs program the ribosome to undergo a noncanonical translation event, translational frameshifting, translational hopping, or termination readthrough. These sequences are termed recoding sites, because they cause the ribosome to change temporarily its coding rules. Cis and trans-acting factors sensitively modulate the efficiency of recoding events. In an attempt to quantitate the effect of these factors we have developed a dual-reporter vector using the lacZ and luc genes to directly measure recoding efficiency. We were able to confirm the effect of several factors that modulate frameshift or readthrough efficiency at a variety of sites. Surprisingly, we were not able to confirm that the complex of factors termed the surveillance complex regulates translational frameshifting. This complex regulates degradation of nonsense codon-containing mRNAs and we confirm that it also affects the efficiency of nonsense suppression. Our data suggest that the surveillance complex is not a general regulator of translational accuracy, but that its role is closely tied to the translational termination and initiation processes.

Functional and genetic analysis of regulatory regions of coliphage H-19B: location of shiga-like toxin and lysis genes suggest a role for phage functions in toxin release.

Analysis of the DNA sequence of a 17 kb region of the coli lambdoid phage H-19B genome located the genes encoding shiga-like toxin I (Stx-I) downstream of the gene encoding the analogue of the phage lambda Q transcription activator with its site of action, qut at the associated pR' late promoter, and upstream of the analogues of lambda genes encoding lysis functions. Functional studies, including measurement of the effect of H-19B Q action on levels of Stx expressed from an H-19B prophage, show that the H-19B Q acts as a transcription activator with its associated pR'(qut) by promoting readthrough of transcription terminators. Another toxin-producing phage, 933W, has the identical Q gene and pR'(qut) upstream of the stx-II genes. The H-19B Q also activates Stx-II expression from a 933W prophage. An ORF in H-19B corresponding to the holin lysis genes of other lambdoid phages differs by having only one instead of the usual two closely spaced translation initiation signals that are thought to contribute to the time of lysis. These observations suggest that stx-I expression can be enhanced by transcription from pR' as well as a model for toxin release through cell lysis mediated by action of phage-encoded lysis functions. Functional studies show that open reading frames (ORFs) and sites in H-19B that resemble components of the N transcription antitermination systems controlling early operons of other lambdoid phages similarly promote antitermination. However, this N-like system differs significantly from those of other lambdoid phages.

In Vivoandin VitroEvidence for an Anti-Rho Activity Induced by the Phage P4 Polarity Suppressor Protein Psu

The polarity suppression (Psu) protein of bacteriophage P4 causes suppression of transcriptional polarity inEscherichia coliby overcoming Rho termination factor activity. Two newpsumutants defective in polarity suppression are described. Thepsu5mutation deletes codons 95–98 from about the middle of the gene, and the mutant protein is inactive. Thepsu6mutation changes Phe169 to Val and encodes a temperature-sensitive protein. Constitutive overexpression ofpsu+from a plasmid prevents colony formation, but overexpression of mutant genes (psu5, psu6) does not, suggesting that Psu disturbs essential host function(s). Rho protein synthesis is enhanced several-fold in cells containing wild-type Psu, due to readthrough at therhoattenuator, while the physical stability of Rho is maintained. As a consequence, Psu-producing cells accumulate significantly more Rho than normal cells, reminiscent of termination-defectiverhomutants. The polarity suppression activity induced by Psu is demonstratedin vitroby the efficient readthrough of Rho-dependent terminators λ tR1and TIS2during coupled transcription–translation. Purified Rho protein restores termination at TIS2when added to Psu-containing reactions but NusG does not. The data support the hypothesis that Psu has or elicits an anti-Rho function.

Transcripts That Increase the Processivity and Elongation Rate of RNA Polymerase

Transcripts encoded by the cis-acting antitermination sites ( put sites) of lambdoid phage HK022 promote readthrough of downstream transcription terminators. Proper conformation of the transcripts is essential for activity, since put mutations that prevent the formation of predicted RNA stems prevented antitermination, and suppressor mutations that restore the stems restored antitermination. Antitermination does not appear to require proteins other than RNA polymerase, since put-dependent readthrough of multiple sequential terminators was observed in a purified transcription system consisting of template, polymerase, substrates, and buffer. Transcription of put also increased the elongation rate of polymerase, very likely by suppressing pausing. A mutation that alters the zinc-finger region of the β′ subunit of polymerase specifically prevented the put-dependent increases in terminator readthrough and elongation rate. The simplicity of HK022 antitermination contrasts with that of other known antitermination pathways. We propose that the central effector is a transcript that directly alters the elongation properties of RNA polymerase.