Leaderless mRNAs in the Spotlight: Ancient but Not Outdated!

Beck, Heather J.; Moll, Isabella

doi:10.1128/9781683670247.ch10

Cited by 13 publications

(23 citation statements)

References 137 publications

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“…In contrast, lmRNA showed the ‘TANNNT’ motif, which is the conserved −10 motif of the promoter as expected (97.9% (424 of 433), P < 0.05; MEME). As the interaction between fMet-tRNA fMet and 5′-AUG is the unique ribosome binding signal without SD-aSD interaction on lmRNA (49), the determinant of differential translation efficiency would be the downstream RNA structure of 5′-AUG. Interestingly, the absolute value of folding free energy of the 40 bp sequence downstream from a start codon was significantly lower for lmRNA than for mmRNA and emRNA (Mann–Whitney U test, ** P < 0.001, two-sided) (Figure 5E). This suggests that lmRNAs have less structured RNA for efficient translation without 5′-UTRs.…”

Section: Resultsmentioning

confidence: 99%

“…lmRNAs were universally observed among broad species, and their proportion was conserved among phylogenetically close species (49). Thus, they are considered as an evolutionarily ancient translation mode due to the lack of translational machinery such as 5′-UTR and r-proteins (49), but also as a regulatory system for some cases such as kasugamycin resistance (56). Neither functional enrichment nor a remarkable trend of RNA, RPF, and RPF/RNA ratios were observed.…”

Section: Discussionmentioning

confidence: 99%

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Primary transcriptome and translatome analysis determines transcriptional and translational regulatory elements encoded in the Streptomyces clavuligerus genome

Hwang

Lee

Jeong

et al. 2019

Nucleic Acids Research

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Determining transcriptional and translational regulatory elements in GC-rich Streptomyces genomes is essential to elucidating the complex regulatory networks that govern secondary metabolite biosynthetic gene cluster (BGC) expression. However, information about such regulatory elements has been limited for Streptomyces genomes. To address this limitation, a high-quality genome sequence of β-lactam antibiotic-producing Streptomyces clavuligerus ATCC 27 064 is completed, which contains 7163 newly annotated genes. This provides a fundamental reference genome sequence to integrate multiple genome-scale data types, including dRNA-Seq, RNA-Seq and ribosome profiling. Data integration results in the precise determination of 2659 transcription start sites which reveal transcriptional and translational regulatory elements, including −10 and −35 promoter components specific to sigma (σ) factors, and 5′-untranslated region as a determinant for translation efficiency regulation. Particularly, sequence analysis of a wide diversity of the −35 components enables us to predict potential σ-factor regulons, along with various spacer lengths between the −10 and −35 elements. At last, the primary transcriptome landscape of the β-lactam biosynthetic pathway is analyzed, suggesting temporal changes in metabolism for the synthesis of secondary metabolites driven by transcriptional regulation. This comprehensive genetic information provides a versatile genetic resource for rational engineering of secondary metabolite BGCs in Streptomyces.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Primary transcriptome and translatome analysis determines transcriptional and translational regulatory elements encoded in the Streptomyces clavuligerus genome

Hwang

Lee

Jeong

et al. 2019

Nucleic Acids Research

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“…In addition, copJ , merP , rpoN , and czcI 2 were transcribed into leaderless mRNAs. The prevalence of leaderless mRNAs has been shown to be higher in organisms with an earlier evolutionary age, and has been linked to extreme habitats [ 127 , 128 ].…”

Section: Resultsmentioning

confidence: 99%

The Transcriptomic Landscape of Cupriavidus metallidurans CH34 Acutely Exposed to Copper

Maertens

Leys

Matroule

2020

Genes

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Bacteria are increasingly used for biotechnological applications such as bioremediation, biorecovery, bioproduction, and biosensing. The development of strains suited for such applications requires a thorough understanding of their behavior, with a key role for their transcriptomic landscape. We present a thorough analysis of the transcriptome of Cupriavidus metallidurans CH34 cells acutely exposed to copper by tagRNA-sequencing. C. metallidurans CH34 is a model organism for metal resistance, and its potential as a biosensor and candidate for metal bioremediation has been demonstrated in multiple studies. Several metabolic pathways were impacted by Cu exposure, and a broad spectrum of metal resistance mechanisms, not limited to copper-specific clusters, was overexpressed. In addition, several gene clusters involved in the oxidative stress response and the cysteine-sulfur metabolism were induced. In total, 7500 transcription start sites (TSSs) were annotated and classified with respect to their location relative to coding sequences (CDSs). Predicted TSSs were used to re-annotate 182 CDSs. The TSSs of 2422 CDSs were detected, and consensus promotor logos were derived. Interestingly, many leaderless messenger RNAs (mRNAs) were found. In addition, many mRNAs were transcribed from multiple alternative TSSs. We observed pervasive intragenic TSSs both in sense and antisense to CDSs. Antisense transcripts were enriched near the 5′ end of mRNAs, indicating a functional role in post-transcriptional regulation. In total, 578 TSSs were detected in intergenic regions, of which 35 were identified as putative small regulatory RNAs. Finally, we provide a detailed analysis of the main copper resistance clusters in CH34, which include many intragenic and antisense transcripts. These results clearly highlight the ubiquity of noncoding transcripts in the CH34 transcriptome, many of which are putatively involved in the regulation of metal resistance.

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“…Importantly, the ORFs initiated at the 74 combined transcription/translation initiation start sites of LL-mRNAs are readily 75 identifiable from transcriptomic data sets and thus provide a high-confidence list of 76 expressed novel mycobacterial sORFs. Insights into the mechanistic and functional 77 attributes of LL-mRNAs have lagged, as they are rare or poorly expressed in E. coli but 78 are abundant in archaea, Actinobacteria and extremophiles (Beck and Moll 2018). LL-79 sORFs represent the first (5'-most) ORF of a transcript, which positions them for a role in 80 cis-regulation of the downstream operonic genes.…”

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confidence: 99%

Polycysteine-encoding leaderless short ORFs function as cysteine-responsive attenuators of operonic gene expression in mycobacteria

Canestrari

Lasek‐Nesselquist

Upadhyay‬

et al. 2019

Preprint

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24 25 Genome-wide transcriptomic analyses have revealed abundant expressed short open 26 reading frames (ORFs) in bacteria. Whether these short ORFs, or the small proteins 27 they encode, are functional remains an open question. One quarter of mycobacterial 28 mRNAs are leaderless, meaning the RNAs begin with a 5'-AUG or GUG initiation 29 codon. Leaderless mRNAs often encode an unannotated short ORF as the first gene 30 of a polycistronic transcript. Consecutive cysteine codons are highly 31 overrepresented in mycobacterial leaderless short ORFs. Here we show that 32 polycysteine-encoding leaderless short ORFs function as cysteine-responsive 33 attenuators of operonic gene expression. Through detailed mutational analysis, we 34 show that one such polycysteine-encoding short ORF controls expression of the 35 downstream genes by causing ribosome stalling under conditions of low cysteine. 36 Ribosome stalling in turn blocks mRNA secondary structures that otherwise 37 sequester the Shine-Dalgarno ribosome-binding site of the 3'gene. This translational 38 attenuation does not require competing transcriptional terminator formation, a 39 mechanism that underlies traditional amino acid attenuation systems. We further 40 assessed cysteine attenuation in Mycobacterium smegmatis using mass spectrometry 41 to evaluate endogenous proteomic responses. Notably, six cysteine metabolic loci 42 that have unannotated polycisteine-encoding leaderless short ORF architectures 43 responded to cysteine supplementation/limitation, indicating that cysteine-44 responsive attenuation is widespread in mycobacteria. Individual leaderless short 45 ORFs confer independent operon-level control, while their shared dependence on 46 cysteine ensures a collective response. Bottom-up regulon coordination is the 47 antithesis of traditional top-down master regulator regulons and illustrates one 48 utility of the many unnanotated short ORFs expressed in bacterial genomes. 49 50 129We next tested whether cysteine-dependent regulation of Ms5788 requires translation of 130 the upstream Ms5788A LL-sORF. We mutated the GUG translation initiation codon of 131Ms5788A to ACC in the context of the luciferase reporter, to prevent ribosome loading. 132This non-start mutation reduced luciferase expression, and abolished attenuation ( Fig 1B 133 compare i vs ii). These data indicate that translation of Ms5788A is required for cysteine-134 dependent regulation of Ms5788, and that in the absence of Ms5788A translation, 135 expression of Ms5788 is locked in an attenuated state. We speculated that ribosome 136

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Leaderless mRNAs in the Spotlight: Ancient but Not Outdated!

Cited by 13 publications

References 137 publications

Primary transcriptome and translatome analysis determines transcriptional and translational regulatory elements encoded in the Streptomyces clavuligerus genome

Primary transcriptome and translatome analysis determines transcriptional and translational regulatory elements encoded in the Streptomyces clavuligerus genome

The Transcriptomic Landscape of Cupriavidus metallidurans CH34 Acutely Exposed to Copper

Polycysteine-encoding leaderless short ORFs function as cysteine-responsive attenuators of operonic gene expression in mycobacteria

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