aCPSF1 cooperates with terminator U-tract to dictate archaeal transcription termination efficacy

Abstract

Recently, aCPSF1 was reported to function as the long-sought global transcription termination factor of archaea; however, the working mechanism remains elusive. This work, through analyzing transcript-3′end-sequencing data of Methanococcus maripaludis, found genome-wide positive correlations of both the terminator uridine(U)-tract and aCPSF1 with hierarchical transcription termination efficacies (TTEs). In vitro assays determined that aCPSF1 specifically binds to the terminator U-tract with U-tract number-related binding affinity, and in vivo assays demonstrated the two elements are indispensable in dictating high TTEs, revealing that aCPSF1 and the terminator U-tract cooperatively determine high TTEs. The N-terminal KH domains equip aCPSF1 with specific-binding capacity to terminator U-tract and the aCPSF1-terminator U-tract cooperation; while the nuclease activity of aCPSF1 was also required for TTEs. aCPSF1 also guarantees the terminations of transcripts with weak intrinsic terminator signals. aCPSF1 orthologs from Lokiarchaeota and Thaumarchaeota exhibited similar U-tract cooperation in dictating TTEs. Therefore, aCPSF1 and the intrinsic U-rich terminator could work in a noteworthy two-in-one termination mode in archaea, which may be widely employed by archaeal phyla; using one trans-action factor to recognize U-rich terminator signal and cleave transcript 3′-end, the archaeal aCPSF1-dependent transcription termination may represent a simplified archetypal mode of the eukaryotic RNA polymerase II termination machinery.

CRISPR-Cas9 Toolkit for Genome Editing in an Autotrophic CO 2 -Fixing Methanogenic Archaeon

“…In situ tagging is necessary for investigating the cellular functions of genes under physiological conditions, where the studied gene is expressed at its in situ condition. Thus, we explored Cas9-based genome editing method to achieve the in situ tagging of two essential genes, those encoding the RNA polymerase subunit L (RpoL) and the recently identified general transcription termination factor aCPSF1 ( 33 , 34 ). Plasmid pMEV4 was modified by changing both the sgRNA and the donor sequences.…”

“…Consequently, we further evaluated the ability of the CRISPR Cas9 system to generate nucleotide substitutions in a transcriptional terminator ( Fig. 6A ), a featured U-tract sequence upstream of the identified transcription termination site (TTS) of MMP0290 ( 33 , 34 ). To introduce point mutations in this terminator, we modified the Cas9-carried pMEV4 by expressing an sgRNA that targeted a 20-nt sequence flanking the TTS of MMP0290 and inserting an ~1-kb DNA sequence flanking the TTS and with it in the center as the donor.…”

“… Cas9-based in situ multinucleotide mutagenesis to determine the key residues in a terminator of M. maripaludis . (A) Design schematic showing the in situ mutagenesis of multiple nucleotides in a terminator identified by Term-seq previously ( 33 , 34 ). An sgRNA that targets the terminator region (orange bar with the asterisk indicating the nucleotide mutation) was expressed, and the multiple nucleotides in blue letters that were designed to replace the U-rich sequence of the terminator were introduced into the donor sequence at the pMEV4 plasmids.…”

“…Its capability of converting CO 2 and H 2 into CH 4 and its fast autotrophic growth on CO 2 have made M. maripaludis an attractive archaeal chassis in inexpensively producing high-value biochemical products, such as hydrogen, methanol, and geraniol ( 16 , 27 ), indicating the high potential of M. maripaludis as a superior CO 2 -fixing cell factory for fundamental and experimental biotechnology research. This species is also regarded as a model archaeon in archaeal biology research, such as in transcription and posttranscription regulation ( 33 – 35 ), motility characteristics ( 36 , 37 ), featured CO 2 and N 2 fixation, and methanogenic metabolism ( 38 – 45 ). By taking advantage of its genetic system, we previously found the long-sought transcription termination factor aCPSF1 and its mediated archaeal termination mechanism, which represents a bacterium-distinct and simplified archetypal mode of eukaryotic RNA polymerase II termination machinery ( 14 , 34 ).…”

“…This species is also regarded as a model archaeon in archaeal biology research, such as in transcription and posttranscription regulation ( 33 – 35 ), motility characteristics ( 36 , 37 ), featured CO 2 and N 2 fixation, and methanogenic metabolism ( 38 – 45 ). By taking advantage of its genetic system, we previously found the long-sought transcription termination factor aCPSF1 and its mediated archaeal termination mechanism, which represents a bacterium-distinct and simplified archetypal mode of eukaryotic RNA polymerase II termination machinery ( 14 , 34 ). However, although the conventional genome editing tools for M. maripaludis have contributed greatly to the knowledge of archaeal biology and potential biotechnological applications, further improvements are warranted to achieve faster, more efficient, and more versatile genome editing, which will enhance M. maripaludis as a model in archaeal biology and a chassis in archaeal biotechnology.…”