The miniature CRISPR-Cas12m effector binds DNA to block transcription.

CRISPR-Cas are prokaryotic adaptive immune systems. Cas nucleases generally use CRISPR-derived RNA guides to specifically bind and cleave DNA or RNA targets. Here, we describe the experimental characterization of a bacterial CRISPR effector protein Cas12m representing subtype V-M. Despite being less than half the size of Cas12a, Cas12m catalyzes auto-processing of a crRNA guide, recognizes a 5'-TTN' protospacer-adjacent motif (PAM), and stably binds a guide-complementary double-stranded DNA (dsDNA). Cas12m has a RuvC domain with a non-canonical catalytic site and accordingly is incapable of guide-dependent cleavage of target nucleic acids. Despite lacking target cleavage activity, the high binding affinity of Cas12m to dsDNA targets allows for interference as demonstrated by its ability to protect bacteria against invading plasmids through silencing invader transcription and/or replication. Based on these molecular features, we repurposed Cas12m by fusing it to a cytidine deaminase that resulted in base editing within a distinct window.

Anti-CRISPR prediction using deep learning reveals an inhibitor of Cas13b nucleases.

As part of the ongoing bacterial-phage arms race, CRISPR-Cas systems in bacteria clear invading phages whereas anti-CRISPR proteins (Acrs) in phages inhibit CRISPR defenses. Known Acrs have proven extremely diverse, complicating their identification. Here, we report a deep learning algorithm for Acr identification that revealed an Acr against type VI-B CRISPR-Cas systems. The algorithm predicted numerous putative Acrs spanning almost all CRISPR-Cas types and subtypes, including over 7,000 putative type IV and VI Acrs not predicted by other algorithms. By performing a cell-free screen for Acr hits against type VI-B systems, we identified a potent inhibitor of Cas13b nucleases we named AcrVIB1. AcrVIB1 blocks Cas13b-mediated defense against a targeted plasmid and lytic phage, and its inhibitory function principally occurs upstream of ribonucleoprotein complex formation. Overall, our work helps expand the known Acr universe, aiding our understanding of the bacteria-phage arms race and the use of Acrs to control CRISPR technologies.

The temperature-regulated DEAD-box RNA helicase CrhR interactome: Autoregulation and photosynthesis-related transcripts.

RNA helicases play crucial functions in RNA biology. In plants, RNA helicases are encoded by large gene families, performing roles in abiotic stress responses, development, the post-transcriptional regulation of gene expression as well as house-keeping functions. Several of these RNA helicases are targeted to the organelles, mitochondria and chloroplasts. Cyanobacteria are the direct evolutionary ancestors of plant chloroplasts. The cyanobacterium Synechocystis 6803 encodes a single DEAD-box RNA helicase, CrhR, that is induced by a range of abiotic stresses, including low temperature. Though the ΔcrhR mutant exhibits a severe cold-sensitive phenotype, the physiological function(s) performed by CrhR have not been described. To identify transcripts interacting with CrhR, we performed RNA co-immunoprecipitation with extracts from a Synechocystis crhR deletion mutant expressing the FLAG-tagged native CrhR or a K57A mutated version with an anticipated enhanced RNA binding. The composition of the interactome was strikingly biased towards photosynthesis-associated and redox-controlled transcripts. A transcript highly enriched in all experiments was the crhR mRNA, suggesting an auto-regulatory molecular mechanism. The identified interactome explains the described physiological role of CrhR in response to the redox poise of the photosynthetic electron transport chain and characterizes CrhR as an enzyme with a diverse range of transcripts as molecular targets.

Similarities and differences between 6S RNAs from Bradyrhizobium japonicum and Sinorhizobium meliloti.

6S RNA, a conserved and abundant small non-coding RNA found in most bacteria, regulates gene expression by inhibiting RNA polymerase (RNAP) holoenzyme. 6S RNAs from α-proteobacteria have been studied poorly so far. Here, we present a first in-depth analysis of 6S RNAs from two α-proteobacteria species, Bradyrhizobium japonicum and Sinorhizobium meliloti. Although both belong to the order Rhizobiales and are typical nitrogen-fixing symbionts of legumes, their 6S RNA expression profiles were found to differ: B. japonicum 6S RNA accumulated in the stationary phase, thus being reminiscent of Escherichia coli 6S RNA, whereas S. meliloti 6S RNA level peaked at the transition to the stationary phase, similarly to Rhodobacter sphaeroides 6S RNA. We demonstrated in vitro that both RNAs have hallmarks of 6S RNAs: they bind to the σ70-type RNAP holoenzyme and serve as templates for de novo transcription of so-called product RNAs (pRNAs) ranging in length from ∼13 to 24 nucleotides, with further evidence of the synthesis of even longer pRNAs. Likewise, stably bound pRNAs were found to rearrange the 6S RNA structure to induce its dissociation from RNAP. Compared with B. japonicum 6S RNA, considerable conformational heterogeneity was observed for S. meliloti 6S RNA and its complexes with pRNAs, even though the two 6S RNAs share ∼75% sequence identity. Overall, our findings suggest that the two rhizobial 6S RNAs have diverged with respect to their regulatory impact on gene expression throughout the bacterial life cycle.

RNA helicase-regulated processing of the Synechocystis rimO-crhR operon results in differential cistron expression and accumulation of two sRNAs.

The arrangement of functionally-related genes in operons is a fundamental element of how genetic information is organized in prokaryotes. This organization ensures coordinated gene expression by co-transcription. Often, however, alternative genetic responses to specific stress conditions demand the discoordination of operon expression. During cold temperature stress, accumulation of the gene encoding the sole Asp-Glu-Ala-Asp (DEAD)-box RNA helicase in Synechocystis sp. PCC 6803, crhR (slr0083), increases 15-fold. Here, we show that crhR is expressed from a dicistronic operon with the methylthiotransferase rimO/miaB (slr0082) gene, followed by rapid processing of the operon transcript into two monocistronic mRNAs. This cleavage event is required for and results in destabilization of the rimO transcript. Results from secondary structure modeling and analysis of RNase E cleavage of the rimO-crhR transcript in vitro suggested that CrhR plays a role in enhancing the rate of the processing in an auto-regulatory manner. Moreover, two putative small RNAs are generated from additional processing, degradation, or both of the rimO transcript. These results suggest a role for the bacterial RNA helicase CrhR in RNase E-dependent mRNA processing in Synechocystis and expand the known range of organisms possessing small RNAs derived from processing of mRNA transcripts.

Inactivation of the RNA helicase CrhR impacts a specific subset of the transcriptome in the cyanobacterium Synechocystis sp. PCC 6803.

DEAD-box RNA-helicases catalyze the reorganization of structured RNAs and the formation of RNP complexes. The cyanobacterium Synechocystis sp. PCC 6803 encodes a single DEAD-box RNA helicase, CrhR (Slr0083), whose expression is regulated by abiotic stresses that alter the redox potential of the photosynthetic electron transport chain, including temperature downshift. Despite its proposed effect on RNA metabolism and its known relevance in cold-stress adaptation, the reported impact of a CrhR knockout on the cold adaption of the transcriptome only identified eight affected genes. Here, we utilized a custom designed microarray to assess the impact of the absence of CrhR RNA helicase activity on the transcriptome, independent of cold stress. CrhR truncation impacts an RNA subset comprising ~10% of the ncRNA and also ~10% of the mRNA transcripts. While equal numbers of mRNAs showed increased as well as decreased abundance, more than 90% of the ncRNAs showed enhanced expression in the absence of CrhR, indicative of a negative effect on ncRNA transcription or stability. We further tested the effect of CrhR on the stability of strongly responding RNAs that identify examples of post-transcriptional and transcriptional regulation. The data suggest that CrhR impacts multiple aspects of RNA metabolism in Synechocystis.

A study on endonuclease BspD6I and its stimulus-responsive switching by modified oligonucleotides.

Nicking endonucleases (NEases) selectively cleave single DNA strands in double-stranded DNAs at a specific site. They are widely used in bioanalytical applications and in genome editing; however, the peculiarities of DNA-protein interactions for most of them are still poorly studied. Previously, it has been shown that the large subunit of heterodimeric restriction endonuclease BspD6I (Nt.BstD6I) acts as a NEase. Here we present a study of interaction of restriction endonuclease BspD6I with modified DNA containing single non-nucleotide insertion with an azobenzene moiety in the enzyme cleavage sites or in positions of sugar-phosphate backbone nearby. According to these data, we designed a number of effective stimulus-responsive oligonucleotide inhibitors bearing azobenzene or triethylene glycol residues. These modified oligonucleotides modulated the functional activity of Nt.BspD6I after cooling or heating. We were able to block the cleavage of T7 phage DNA by this enzyme in the presence of such inhibitors at 20-25°C, whereas the Nt.BspD6I ability to hydrolyze DNA was completely restored after heating to 45°C. The observed effects can serve as a basis for the development of a platform for regulation of NEase activity in vitro or in vivo by external signals.

Conserved small mRNA with an unique, extended Shine-Dalgarno sequence.

Up to now, very small protein-coding genes have remained unrecognized in sequenced genomes. We identified an mRNA of 165 nucleotides (nt), which is conserved in Bradyrhizobiaceae and encodes a polypeptide with 14 amino acid residues (aa). The small mRNA harboring a unique Shine-Dalgarno sequence (SD) with a length of 17 nt was localized predominantly in the ribosome-containing P100 fraction of Bradyrhizobium japonicum USDA 110. Strong interaction between the mRNA and 30S ribosomal subunits was demonstrated by their co-sedimentation in sucrose density gradient. Using translational fusions with egfp, we detected weak translation and found that it is impeded by both the extended SD and the GTG start codon (instead of ATG). Biophysical characterization (CD- and NMR-spectroscopy) showed that synthesized polypeptide remained unstructured in physiological puffer. Replacement of the start codon by a stop codon increased the stability of the transcript, strongly suggesting additional posttranscriptional regulation at the ribosome. Therefore, the small gene was named rreB (ribosome-regulated expression in Bradyrhizobiaceae). Assuming that the unique ribosome binding site (RBS) is a hallmark of rreB homologs or similarly regulated genes, we looked for similar putative RBS in bacterial genomes and detected regions with at least 16 nt complementarity to the 3'-end of 16S rRNA upstream of sORFs in Caulobacterales, Rhizobiales, Rhodobacterales and Rhodospirillales. In the Rhodobacter/Roseobacter lineage of α-proteobacteria the corresponding gene (rreR) is conserved and encodes an 18 aa protein. This shows how specific RBS features can be used to identify new genes with presumably similar control of expression at the RNA level.

Peculiarities of the interaction of the restriction endonuclease BspD6I with DNA containing its recognition site.

BACKGROUND: Nicking endonucleases are enzymes that recognize specific sites in double-stranded DNA and cleave only one strand at a predetermined position. These enzymes are involved in DNA replication and repair; they can also function as subunits of bacterial heterodimeric restriction endonucleases. One example of such a proteins is the restriction endonuclease BspD6I (R.BspD6I) from Bacillus species strain D6, which consists of the large subunit - nicking endonuclease BspD6I (Nt.BspD6I), and the small subunit (ss.BspD6I). Nt.BspD6I can function independently. Similar enzymes are now widely used in numerous biotechnological applications. The aim of this study was to investigate the fundamental properties of two subunits of R.BspD6I and their interdependence in the course of R.BspD6I activity. METHODS: The binding and hydrolysis of DNA duplexes by R.BspD6I are primary analyzed by gel electrophoresis. To elucidate the difference between Nt.BspD6I interaction with the substrate and product of hydrolysis, the thickness shear mode acoustic method is used. RESULTS AND CONCLUSIONS: The thermodynamic and kinetic parameters of the Nt.BspD6I interaction with DNA are determined. For the first time we demonstrated that Nt.BspD6I bends the DNA during complex formation. Nt.BspD6I is able to form complexes with the product nicked in the top strand and ss.BspD6I cleaves the bottom strand of the DNA consecutively. Furthermore, the influence of dA methylation in the R.BspD6I recognition site on ss.BspD6I activity is analyzed. GENERAL SIGNIFICANCE: The obtained results provide evidence that Nt.BspD6I coordinates the activity of R.BspD6I by strictly coupling of the bottom strand cleavage by ss.BspD6I to the top strand cleavage.

Thermo-switchable activity of the restriction endonuclease SsoII achieved by site-directed enzyme modification.

In this work, the possibility of constructing a thermo-switchable enzyme according to the "molecular gate" strategy is demonstrated. The approach is based on the covalent attachment of oligodeoxyribonucleotides to cysteine residues of an enzyme adjacent to its active center to form a temporal barrier for enzyme-substrate complex formation. The activity of the modified enzyme that had been studied here-the restriction endonuclease SsoII (R.SsoII)-was diminished by a factor of 180 at 25 °Ð¡ that almost abolished the enzymatic activity when compared with the unmodified enzyme. However, heating of the modified enzyme to 45 °Ð¡ resulted in a 30-fold increase of activity. The activity of unmodified R.SsoII also increased on heating from 25 to 45 °; however, the difference did not exceed a factor of 3-4. The changes in enzymatic activity observed were shown to be reversible for both the unmodified and the modified R.SsoII. Variation of the length and the sequence of the attached oligodeoxyribonucleotides might allow greater modulation of the activity of DNA-protein conjugates.