CRISPR interference and priming varies with individual spacer sequences

CRISPR–Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems allow bacteria to adapt to infection by acquiring ‘spacer’ sequences from invader DNA into genomic CRISPR loci. Cas proteins use RNAs derived from these loci to target cognate sequences for destruction through CRISPR interference. Mutations in the protospacer adjacent motif (PAM) and seed regions block interference but promote rapid ‘primed’ adaptation. Here, we use multiple spacer sequences to reexamine the PAM and seed sequence requirements for interference and priming in the Escherichia coli Type I-E CRISPR–Cas system. Surprisingly, CRISPR interference is far more tolerant of mutations in the seed and the PAM than previously reported, and this mutational tolerance, as well as priming activity, is highly dependent on spacer sequence. We identify a large number of functional PAMs that can promote interference, priming or both activities, depending on the associated spacer sequence. Functional PAMs are preferentially acquired during unprimed ‘naïve’ adaptation, leading to a rapid priming response following infection. Our results provide numerous insights into the importance of both spacer and target sequences for interference and priming, and reveal that priming is a major pathway for adaptation during initial infection

Extensive mis-splicing of a bi-partite plant mitochondrial group II intron

Expression of the seed plant mitochondrial nad5 gene involves two trans-splicing events that remove fragmented group II introns and join the small, central exon c to exons b and d. We show that in both monocot and eudicot plants, extensive mis-splicing of the bi-partite intron 2 takes place, resulting in the formation of aberrantly spliced products in which exon c is joined to various sites within exon b. These mis-spliced products accumulate to levels comparable to or greater than that of the correctly spliced mRNA. We suggest that mis-splicing may result from folding constraints imposed on intron 2 by base-pairing between exon a and a portion of the bi-partite intron 3 downstream of exon c. Consistent with this hypothesis, we find that mis-splicing does not occur in Oenothera mitochondria, where intron 3 is further fragmented such that the predicted base-pairing region is not covalently linked to exon c. Our findings suggest that intron fragmentation may lead to mis-splicing, which may be corrected by further intron fragmentation

Solution structure of a let-7 miRNA:lin-41 mRNA complex from C. elegans

let-7 microRNA (miRNA) regulates heterochronic genes in developmental timing of the nematode Caenorhabditis elegans. Binding of miRNA to messenger RNA (mRNA) and structural features of the complex are crucial for gene silencing. We herein present the NMR solution structure of a model mimicking the interaction of let-7 miRNA with its complementary site (LCS 2) in the 3′ untranslated region (3′-UTR) of the lin-41 mRNA. A structural study was performed by NMR spectroscopy using NOE restraints, torsion angle restraints and residual dipolar couplings. The 33-nt RNA construct folds into a stem–loop structure that features two stem regions which are separated by an asymmetric internal loop. One of the stems comprises a GU wobble base pair, which does not alter its overall A-form RNA conformation. The asymmetric internal loop adopts a single, well-defined structure in which three uracils form a base triple, while two adenines form a base pair. The 3D structure of the construct gives insight into the structural aspects of interactions between let-7 miRNA and lin-41 mRNA

Ligand-induced folding of the thiM TPP riboswitch investigated by a structure-based fluorescence spectroscopic approach

Riboswitches are genetic control elements within non-coding regions of mRNA. They consist of a metabolite-sensitive aptamer and an adjoining expression platform. Here, we describe ligand-induced folding of a thiamine pyrophosphate (TPP) responsive riboswitch from Escherichia coli thiM mRNA, using chemically labeled variants. Referring to a recent structure determination of the TPP/aptamer complex, each variant was synthesized with a single 2-aminopurine (AP) nucleobase replacement that was selected to monitor formation of tertiary interactions of a particular region during ligand binding in real time by fluorescence experiments. We have determined the rate constants for conformational adjustment of the individual AP sensors. From the 7-fold differentiation of these constants, it can be deduced that tertiary contacts between the two parallel helical domains (P2/J3-2/P3/L3 and P4/P5/L5) that grip the ligand's ends in two separate pockets, form significantly faster than the function-critical three-way junction with stem P1 fully developed. Based on these data, we characterize the process of ligand binding by an induced fit of the RNA and propose a folding model of the TPP riboswitch aptamer. For the full-length riboswitch domain and for shorter constructs that represent transcriptional intermediates, we have additionally evaluated ligand-induced folding via AP-modified variants and provide insights into the sequential folding pathway that involves a finely balanced equilibrium of secondary structures

Measuring the dynamic surface accessibility of RNA with the small paramagnetic molecule TEMPOL

The surface accessibility of macromolecules plays a key role in modulating molecular recognition events. RNA is a complex and dynamic molecule involved in many aspects of gene expression. However, there are few experimental methods available to measure the accessible surface of RNA. Here, we investigate the accessible surface of RNA using NMR and the small paramagnetic molecule TEMPOL. We investigated two RNAs with known structures, one that is extremely stable and one that is dynamic. For helical regions, the TEMPOL probing data correlate well with the predicted RNA surface, and the method is able to distinguish subtle variations in atom depths, such as the relative accessibility of pyrimidine versus purine aromatic carbon atoms. Dynamic motions are also detected by TEMPOL probing, and the method accurately reports a previously characterized pH-dependent conformational transition involving formation of a protonated C–A pair and base flipping. Some loop regions are observed to exhibit anomalously high accessibility, reflective of motions that are not evident within the ensemble of NMR structures. We conclude that TEMPOL probing can provide valuable insights into the surface accessibility and dynamics of RNA, and can also be used as an independent means of validating RNA structure and dynamics in solution

Evolution of protein-coupled RNA dynamics during hierarchical assembly of ribosomal complexes

Assembly of 30S ribosomes involves the hierarchical addition of ribosomal proteins that progressively stabilize the folded 16S rRNA. Here, we use three-color single molecule FRET to show how combinations of ribosomal proteins uS4, uS17 and bS20 in the 16S 5' domain enable the recruitment of protein bS16, the next protein to join the complex. Analysis of real-time bS16 binding events shows that bS16 binds both native and non-native forms of the rRNA. The native rRNA conformation is increasingly favored after bS16 binds, explaining how bS16 drives later steps of 30S assembly. Chemical footprinting and molecular dynamics simulations show that each ribosomal protein switches the 16S conformation and dampens fluctuations at the interface between rRNA subdomains where bS16 binds. The results suggest that specific protein-induced changes in the rRNA dynamics underlie the hierarchy of 30S assembly and simplify the search for the native ribosome structure

Cas3 is a limiting factor for CRISPR-Cas immunity in Escherichia coli cells lacking H-NS

Background: CRISPR-Cas systems provide adaptive immunity to mobile genetic elements in prokaryotes. In many bacteria, including E. coli, a specialized ribonucleoprotein complex called Cascade enacts immunity by “an interference reaction" between CRISPR encoded RNA (crRNA) and invader DNA sequences called “protospacers”. Cascade recognizes invader DNA via short “protospacer adjacent motif” (PAM) sequences and crRNA-DNA complementarity. This triggers degradation of invader DNA by Cas3 protein and in some circumstances stimulates capture of new invader DNA protospacers for incorporation into CRISPR as “spacers” by Cas1 and Cas2 proteins, thus enhancing immunity. Co-expression of Cascade, Cas3 and crRNA is effective at giving E. coli cells resistance to phage lysis, if a transcriptional repressor of Cascade and CRISPR, H-NS, is inactivated (Δhns). We present further genetic analyses of the regulation of CRISPR-Cas mediated phage resistance in Δhns E. coli cells. Results: We observed that E. coli Type I-E CRISPR-Cas mediated resistance to phage λ was strongly temperature dependent, when repeating previously published experimental procedures. Further genetic analyses highlighted the importance of culture conditions for controlling the extent of CRISPR immunity in E. coli. These data identified that expression levels of cas3 is an important limiting factor for successful resistance to phage. Significantly, we describe the new identification that cas3 is also under transcriptional control by H-NS but that this is exerted only in stationary phase cells. Conclusions: Regulation of cas3 is responsive to phase of growth, and to growth temperature in E. coli, impacting on the efficacy of CRISPR-Cas immunity in these experimental systems

Impact of Small Repeat Sequences on Bacterial Genome Evolution

Intergenic regions of prokaryotic genomes carry multiple copies of terminal inverted repeat (TIR) sequences, the nonautonomous miniature inverted-repeat transposable element (MITE). In addition, there are the repetitive extragenic palindromic (REP) sequences that fold into a small stem loop rich in G–C bonding. And the clustered regularly interspaced short palindromic repeats (CRISPRs) display similar small stem loops but are an integral part of a complex genetic element. Other classes of repeats such as the REP2 element do not have TIRs but show other signatures. With the current availability of a large number of whole-genome sequences, many new repeat elements have been discovered. These sequences display diverse properties. Some show an intimate linkage to integrons, and at least one encodes a small RNA. Many repeats are found fused with chromosomal open reading frames, and some are located within protein coding sequences. Small repeat units appear to work hand in hand with the transcriptional and/or post-transcriptional apparatus of the cell. Functionally, they are multifaceted, and this can range from the control of gene expression, the facilitation of host/pathogen interactions, or stimulation of the mammalian immune system. The CRISPR complex displays dramatic functions such as an acquired immune system that defends against invading viruses and plasmids. Evolutionarily, mobile repeat elements may have influenced a cycle of active versus inactive genes in ancestral organisms, and some repeats are concentrated in regions of the chromosome where there is significant genomic plasticity. Changes in the abundance of genomic repeats during the evolution of an organism may have resulted in a benefit to the cell or posed a disadvantage, and some present day species may reflect a purification process. The diverse structure, eclectic functions, and evolutionary aspects of repeat elements are described