Unique Archaeal Small RNAs.

Advances in genome-wide sequence technologies allow for detailed insights into the complexity of RNA landscapes of organisms from all three domains of life. Recent analyses of archaeal transcriptomes identified interaction and regulation networks of noncoding RNAs in this understudied domain. Here, we review current knowledge of small, noncoding RNAs with important functions for the archaeal lifestyle, which often requires adaptation to extreme environments. One focus is RNA metabolism at elevated temperatures in hyperthermophilic archaea, which reveals elevated amounts of RNA-guided RNA modification and virus defense strategies. Genome rearrangement events result in unique fragmentation patterns of noncoding RNA genes that require elaborate maturation pathways to yield functional transcripts. RNA-binding proteins, e.g., L7Ae and LSm, are important for many posttranscriptional control functions of RNA molecules in archaeal cells. We also discuss recent insights into the regulatory potential of their noncoding RNA partners.

Structural Variation of Type I-F CRISPR RNA Guided DNA Surveillance.

CRISPR-Cas systems are prokaryotic immune systems against invading nucleic acids. Type I CRISPR-Cas systems employ highly diverse, multi-subunit surveillance Cascade complexes that facilitate duplex formation between crRNA and complementary target DNA for R-loop formation, retention, and DNA degradation by the subsequently recruited nuclease Cas3. Typically, the large subunit recognizes bona fide targets through the PAM (protospacer adjacent motif), and the small subunit guides the non-target DNA strand. Here, we present the Apo- and target-DNA-bound structures of the I-Fv (type I-F variant) Cascade lacking the small and large subunits. Large and small subunits are functionally replaced by the 5' terminal crRNA cap Cas5fv and the backbone protein Cas7fv, respectively. Cas5fv facilitates PAM recognition from the DNA major groove site, in contrast to all other described type I systems. Comparison of the type I-Fv Cascade with an anti-CRISPR protein-bound I-F Cascade reveals that the type I-Fv structure differs substantially at known anti-CRISPR protein target sites and might therefore be resistant to viral Cascade interception.

Anti-CRISPR AcrIF9 functions by inducing the CRISPR-Cas complex to bind DNA non-specifically.

Phages and other mobile genetic elements express anti-CRISPR proteins (Acrs) to protect their genomes from destruction by CRISPR-Cas systems. Acrs usually block the ability of CRISPR-Cas systems to bind or cleave their nucleic acid substrates. Here, we investigate an unusual Acr, AcrIF9, that induces a gain-of-function to a type I-F CRISPR-Cas (Csy) complex, causing it to bind strongly to DNA that lacks both a PAM sequence and sequence complementarity. We show that specific and non-specific dsDNA compete for the same site on the Csy:AcrIF9 complex with rapid exchange, but specific ssDNA appears to still bind through complementarity to the CRISPR RNA. Induction of non-specific DNA-binding is a shared property of diverse AcrIF9 homologues. Substitution of a conserved positively charged surface on AcrIF9 abrogated non-specific dsDNA-binding of the Csy:AcrIF9 complex, but specific dsDNA binding was maintained. AcrIF9 mutants with impaired non-specific dsDNA binding activity in vitro displayed a reduced ability to inhibit CRISPR-Cas activity in vivo. We conclude that misdirecting the CRISPR-Cas complex to bind non-specific DNA is a key component of the inhibitory mechanism of AcrIF9. This inhibitory mechanism is distinct from a previously characterized anti-CRISPR, AcrIF1, that sterically blocks DNA-binding, even though AcrIF1and AcrIF9 bind to the same site on the Csy complex.

Type IV CRISPR-Cas systems are highly diverse and involved in competition between plasmids.

CRISPR-Cas systems provide prokaryotes with adaptive immune functions against viruses and other genetic parasites. In contrast to all other types of CRISPR-Cas systems, type IV has remained largely overlooked. Here, we describe a previously uncharted diversity of type IV gene cassettes, primarily encoded by plasmid-like elements from diverse prokaryotic taxa. Remarkably, via a comprehensive analysis of their CRISPR spacer content, these systems were found to exhibit a strong bias towards the targeting of other plasmids. Our data indicate that the functions of type IV systems have diverged from those of other host-related CRISPR-Cas immune systems to adopt a role in mediating conflicts between plasmids. Furthermore, we find evidence for cross-talk between certain type IV and type I CRISPR-Cas systems that co-exist intracellularly, thus providing a simple answer to the enigmatic absence of type IV adaptation modules. Collectively, our results lead to the expansion and reclassification of type IV systems and provide novel insights into the biological function and evolution of these elusive systems.

A regulatory RNA is involved in RNA duplex formation and biofilm regulation in Sulfolobus acidocaldarius.

Non-coding RNAs (ncRNA) are involved in essential biological processes in all three domains of life. The regulatory potential of ncRNAs in Archaea is, however, not fully explored. In this study, RNA-seq analyses identified a set of 29 ncRNA transcripts in the hyperthermophilic archaeon Sulfolobus acidocaldarius that were differentially expressed in response to biofilm formation. The most abundant ncRNA of this set was found to be resistant to RNase R treatment (RNase R resistant RNA, RrrR(+)) due to duplex formation with a reverse complementary RNA (RrrR(-)). The deletion of the RrrR(+) gene resulted in significantly impaired biofilm formation, while its overproduction increased biofilm yield. RrrR(+) was found to act as an antisense RNA against the mRNA of a hypothetical membrane protein. The RrrR(+) transcript was shown to be stabilized by the presence of the RrrR(-) strand in S. acidocaldarius cell extracts. The accumulation of these RrrR duplexes correlates with an apparent absence of dsRNA degrading RNase III domains in archaeal proteins.

tRNA Modification Profiles and Codon-Decoding Strategies in Methanocaldococcus jannaschii.

tRNAs play a critical role in mRNA decoding, and posttranscriptional modifications within tRNAs drive decoding efficiency and accuracy. The types and positions of tRNA modifications in model bacteria have been extensively studied, and tRNA modifications in a few eukaryotic organisms have also been characterized and localized to particular tRNA sequences. However, far less is known regarding tRNA modifications in archaea. While the identities of modifications have been determined for multiple archaeal organisms, Haloferax volcanii is the only organism for which modifications have been extensively localized to specific tRNA sequences. To improve our understanding of archaeal tRNA modification patterns and codon-decoding strategies, we have used liquid chromatography and tandem mass spectrometry to characterize and then map posttranscriptional modifications on 34 of the 35 unique tRNA sequences of Methanocaldococcus jannaschii A new posttranscriptionally modified nucleoside, 5-cyanomethyl-2-thiouridine (cnm5s2U), was discovered and localized to position 34. Moreover, data consistent with wyosine pathway modifications were obtained beyond the canonical tRNAPhe as is typical for eukaryotes. The high-quality mapping of tRNA anticodon loops enriches our understanding of archaeal tRNA modification profiles and decoding strategies.IMPORTANCE While many posttranscriptional modifications in M. jannaschii tRNAs are also found in bacteria and eukaryotes, several that are unique to archaea were identified. By RNA modification mapping, the modification profiles of M. jannaschii tRNA anticodon loops were characterized, allowing a comparative analysis with H. volcanii modification profiles as well as a general comparison with bacterial and eukaryotic decoding strategies. This general comparison reveals that M. jannaschii, like H. volcanii, follows codon-decoding strategies similar to those used by bacteria, although position 37 appears to be modified to a greater extent than seen in H. volcanii.

PAM identification by CRISPR-Cas effector complexes: diversified mechanisms and structures.

Adaptive immunity of prokaryotes is mediated by CRISPR-Cas systems that employ a large variety of Cas protein effectors to identify and destroy foreign genetic material. The different targeting mechanisms of Cas proteins rely on the proper protection of the host genome sequence while allowing for efficient detection of target sequences, termed protospacers. A short DNA sequence, the protospacer-adjacent motif (PAM), is frequently used to mark proper target sites. Cas proteins have evolved a multitude of PAM-interacting domains, which enables them to cope with viral anti-CRISPR measures that alter the sequence or accessibility of PAM elements. In this review, we summarize known PAM recognition strategies for all CRISPR-Cas types. Available structures of target bound Cas protein effector complexes highlight the diversity of mechanisms and domain architectures that are employed to guarantee target specificity.

Plasticity of archaeal C/D box sRNA biogenesis.

Archaeal and eukaryotic organisms contain sets of C/D box s(no)RNAs with guide sequences that determine ribose 2'-O-methylation sites of target RNAs. The composition of these C/D box sRNA sets is highly variable between organisms and results in varying RNA modification patterns which are important for ribosomal RNA folding and stability. Little is known about the genomic organization of C/D box sRNA genes in archaea. Here, we aimed to obtain first insights into the biogenesis of these archaeal C/D box sRNAs and analyzed the genetic context of more than 300 archaeal sRNA genes. We found that the majority of these genes do not possess independent promoters but are rather located at positions that allow for co-transcription with neighboring genes and their start or stop codons were frequently incorporated into the conserved boxC and D motifs. The biogenesis of plasmid-encoded C/D box sRNA variants was analyzed in vivo in Sulfolobus acidocaldarius. It was found that C/D box sRNA maturation occurs independent of their genetic context and relies solely on the presence of intact RNA kink-turn structures. The observed plasticity of C/D box sRNA biogenesis is suggested to enable their accelerated evolution and, consequently, allow for adjustments of the RNA modification landscape.

Modulating the Cascade architecture of a minimal Type I-F CRISPR-Cas system.

Shewanella putrefaciens CN-32 contains a single Type I-Fv CRISPR-Cas system which confers adaptive immunity against bacteriophage infection. Three Cas proteins (Cas6f, Cas7fv, Cas5fv) and mature CRISPR RNAs were shown to be required for the assembly of an interference complex termed Cascade. The Cas protein-CRISPR RNA interaction sites within this complex were identified via mass spectrometry. Additional Cas proteins, commonly described as large and small subunits, that are present in all other investigated Cascade structures, were not detected. We introduced this minimal Type I system in Escherichia coli and show that it provides heterologous protection against lambda phage. The absence of a large subunit suggests that the length of the crRNA might not be fixed and recombinant Cascade complexes with drastically shortened and elongated crRNAs were engineered. Size-exclusion chromatography and small-angle X-ray scattering analyses revealed that the number of Cas7fv backbone subunits is adjusted in these shortened and extended Cascade variants. Larger Cascade complexes can still confer immunity against lambda phage infection in E. coli Minimized Type I CRISPR-Cas systems expand our understanding of the evolution of Cascade assembly and diversity. Their adjustable crRNA length opens the possibility for customizing target DNA specificity.

Fragmentation of the CRISPR-Cas Type I-B signature protein Cas8b.

BACKGROUND: CRISPR arrays are transcribed into long precursor RNA species, which are further processed into mature CRISPR RNAs (crRNAs). Cas proteins utilize these crRNAs, which contain spacer sequences that can be derived from mobile genetic elements, to mediate immunity during a reoccurring virus infection. Type I CRISPR-Cas systems are defined by the presence of different Cascade interference complexes containing large and small subunits that play major roles during target DNA selection. METHODS: Here, we produce the protein and crRNA components of the Type I-B CRISPR-Cas complex of Clostridium thermocellum and Methanococcus maripaludis. The C. thermocellum Cascade complexes were reconstituted and analyzed via size-exclusion chromatography. Activity of the heterologous M. maripaludis CRISPR-Cas system was followed using phage lambda plaques assays. RESULTS: The reconstituted Type-I-B Cascade complex contains Cas7, Cas5, Cas6b and the large subunit Cas8b. Cas6b can be omitted from the reconstitution protocol. The large subunit Cas8b was found to be represented by two tightly associated protein fragments and a small C-terminal Cas8b segment was identified in recombinant complexes and C. thermocellum cell lysate. CONCLUSIONS: Production of Cas8b generates a small C-terminal fragment, which is suggested to fulfill the role of the missing small subunit. A heterologous, synthetic M. maripaludis Type I-B system is active in E. coli against phage lambda, highlighting a potential for genome editing using endogenous Type-I-B CRISPR-Cas machineries. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.