scholarly journals Identification of RNA 3’ ends and termination sites in Haloferax volcanii

2019 ◽  
Author(s):  
Sarah J. Berkemer ◽  
Lisa-Katharina Maier ◽  
Fabian Amman ◽  
Stephan H. Bernhart ◽  
Julia Wörtz ◽  
...  
Keyword(s):  
Untranslated Region ◽  
Transcription Termination ◽  
Haloferax Volcanii ◽  
Systematic Analysis ◽  
Coding Regions ◽  
Genome Wide ◽  
Rnaseq Data ◽  
Sequence Signature ◽  
Novel Algorithm

AbstractArchaeal genomes are densely packed; thus, correct transcription termination is an important factor for orchestrated gene expression. A systematic analysis of RNA 3’ termini, to identify transcription termination sites (TTS) using RNAseq data has hitherto only been performed in two archaea. In this study, only part of the genome had been investigated. Here, we developed a novel algorithm that allows an unbiased, genome-wide identification of RNA 3’ termini independent of annotation. In an RNA fraction enriched for primary transcripts by terminator exonuclease (TEX) treatment we identified 1,543 RNA 3’ termini. A strong sequence signature consistent with known termination events at intergenic loci indicates a clear enrichment for native TTS among them. Using these data we determined distinct putative termination motifs for intergenic (a T stretch) and coding regions (AGATC). In vivo reporter gene tests of selected TTS confirmed termination at these sites, which exemplify the different motifs. For several genes, more than one termination site was detected, resulting in transcripts with different lengths of the 3’ untranslated region.

3'end maturation of the Chlamydomonas reinhardtii chloroplast atpB mRNA is a two-step process

1993 ◽  
Vol 13 (4) ◽  
pp. 2277-2285
Author(s):  
D B Stern ◽  
K L Kindle
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Higher Plants ◽  
Transcription Termination ◽  
Protein Coding ◽  
Coding Regions ◽  
In Vitro Processing ◽  
Mrna Precursor ◽  
Chloroplast Transcripts

Inverted repeat (IR) sequences are found at the 3' ends of most chloroplast protein coding regions, and we have previously shown that the 3'IR is important for accumulation of atpB mRNA in Chlamydomonas reinhardtii (D. B. Stern, E.R. Radwanski, and K. L. Kindle, Plant Cell 3:285-297, 1991). In vitro studies indicate that 3' IRs are inefficient transcription termination signals in higher plants and have furthermore defined processing activities that act on the 3' ends of chloroplast transcripts, suggesting that most chloroplast mRNAs are processed at their 3' ends in vivo. To investigate the mechanism of 3' end processing in Chlamydomonas reinhardtii chloroplasts, the maturation of atpB mRNA was examined in vitro and in vivo. In vitro, a synthetic atpB mRNA precursor is rapidly cleaved at a position 10 nucleotides downstream from the mature 3' terminus. This cleavage is followed by exonucleolytic processing to generate the mature 3' end. In vivo run-on transcription experiments indicate that a maximum of 50% of atpB transcripts are transcriptionally terminated at or near the IR, while the remainder are subject to 3' end processing. Analysis of transcripts derived from chimeric atpB genes introduced into Chlamydomonas chloroplasts by biolistic transformation suggests that in vivo processing and in vitro processing occur by similar or identical mechanisms.

scholarly journals The conserved ribonuclease aCPSF1 triggers genome-wide transcription termination of Archaea via a 3′-end cleavage mode

2020 ◽  
Vol 48 (17) ◽  
pp. 9589-9605 ◽  
Author(s):  
Lei Yue ◽  
Jie Li ◽  
Bing Zhang ◽  
Lei Qi ◽  
Zhihua Li ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Termination ◽  
Methanococcus Maripaludis ◽  
Genome Wide ◽  
Transcription Termination Factor ◽  
A Genome ◽  
Archaeal Transcription ◽  
Archaeal Ancestor

Abstract Transcription termination defines accurate transcript 3′-ends and ensures programmed transcriptomes, making it critical to life. However, transcription termination mechanisms remain largely unknown in Archaea. Here, we reported the physiological significance of the newly identified general transcription termination factor of Archaea, the ribonuclease aCPSF1, and elucidated its 3′-end cleavage triggered termination mechanism. The depletion of Mmp-aCPSF1 in Methanococcus maripaludis caused a genome-wide transcription termination defect and disordered transcriptome. Transcript-3′end-sequencing revealed that transcriptions primarily terminate downstream of a uridine-rich motif where Mmp-aCPSF1 performed an endoribonucleolytic cleavage, and the endoribonuclease activity was determined to be essential to the in vivo transcription termination. Co-immunoprecipitation and chromatin-immunoprecipitation detected interactions of Mmp-aCPSF1 with RNA polymerase and chromosome. Phylogenetic analysis revealed that the aCPSF1 orthologs are ubiquitously distributed among the archaeal phyla, and two aCPSF1 orthologs from Lokiarchaeota and Thaumarchaeota could replace Mmp-aCPSF1 to terminate transcription of M. maripaludis. Therefore, the aCPSF1 dependent termination mechanism could be widely employed in Archaea, including Lokiarchaeota belonging to Asgard Archaea, the postulated archaeal ancestor of Eukaryotes. Strikingly, aCPSF1-dependent archaeal transcription termination reported here exposes a similar 3′-cleavage mode as the eukaryotic RNA polymerase II termination, thus would shed lights on understanding the evolutionary linking between archaeal and eukaryotic termination machineries.

scholarly journals 3'end maturation of the Chlamydomonas reinhardtii chloroplast atpB mRNA is a two-step process.

1993 ◽  
Vol 13 (4) ◽  
pp. 2277-2285 ◽  
Author(s):  
D B Stern ◽  
K L Kindle
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Higher Plants ◽  
Transcription Termination ◽  
Protein Coding ◽  
Coding Regions ◽  
In Vitro Processing ◽  
Mrna Precursor ◽  
Chloroplast Transcripts

Inverted repeat (IR) sequences are found at the 3' ends of most chloroplast protein coding regions, and we have previously shown that the 3'IR is important for accumulation of atpB mRNA in Chlamydomonas reinhardtii (D. B. Stern, E.R. Radwanski, and K. L. Kindle, Plant Cell 3:285-297, 1991). In vitro studies indicate that 3' IRs are inefficient transcription termination signals in higher plants and have furthermore defined processing activities that act on the 3' ends of chloroplast transcripts, suggesting that most chloroplast mRNAs are processed at their 3' ends in vivo. To investigate the mechanism of 3' end processing in Chlamydomonas reinhardtii chloroplasts, the maturation of atpB mRNA was examined in vitro and in vivo. In vitro, a synthetic atpB mRNA precursor is rapidly cleaved at a position 10 nucleotides downstream from the mature 3' terminus. This cleavage is followed by exonucleolytic processing to generate the mature 3' end. In vivo run-on transcription experiments indicate that a maximum of 50% of atpB transcripts are transcriptionally terminated at or near the IR, while the remainder are subject to 3' end processing. Analysis of transcripts derived from chimeric atpB genes introduced into Chlamydomonas chloroplasts by biolistic transformation suggests that in vivo processing and in vitro processing occur by similar or identical mechanisms.

scholarly journals aCPSF1 controlled archaeal transcription termination: a prototypical eukaryotic model

2019 ◽  
Author(s):  
Lei Yue ◽  
Jie Li ◽  
Bing Zhang ◽  
Lei Qi ◽  
Fangqing Zhao ◽  
...  
Keyword(s):  
Transcription Termination ◽  
Super Resolution ◽  
Microscopy Imaging ◽  
Genome Wide ◽  
Transcription Termination Factor ◽  
A Genome ◽  
Cold Sensitive ◽  
Archaeal Transcription ◽  
Rnap Ii

AbstractTranscription termination defines RNA 3′-ends and guarantees programmed transcriptomes, thus is an essential biological process for life. However, transcription termination mechanisms remain almost unknown in Archaea. Here reported the first general transcription termination factor of Archaea, the conserved ribonuclease aCPSF1, and elucidated its 3′-end cleavage dependent termination mechanism. Depletion of Mmp-aCPSF1 in a methanoarchaeon Methanococcus maripaludis caused a genome-wide transcription termination defect and overall transcriptome chaos, and cold-sensitive growth. Transcript-3′end-sequencing (Term-seq) revealed transcriptions mostly terminated downstream of a uridine-rich terminator motif, where Mmp-aCPSF1 performed cleavage. The endoribonuclease activity was determined essential to terminate transcription in vivo as well. Through super-resolution photoactivated localization microscopy imaging, co-immunoprecipitation, and chromatin immunoprecipitation, we demonstrated that Mmp-aCPSF1 localizes within nucleoid and associates with RNAP and chromosomes. aCPSF1 appears to co-evolve with archaeal RNAPs, and two distant orthologs each from Lokiarchaeota and Thaumarchaeota could replace Mmp-aCPSF1 to termination transcription. Thus, aCPSF1 dependent termination mechanism could be universally employed in Archaea, including Lokiarchaeota, one supposed archaeal ancestor of Eukaryotes. Therefore, the reported aCPSF1 cleavage-dependent termination mode not only hints an archetype of Eukaryotic 3′-end processing/cleavage triggered RNAP II termination, but also would shed lights on understanding the complex eukaryotic termination based on the simplified archaeal model.

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