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CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III

Elitza Deltcheva, Krzysztof Chylinski, Cynthia M. Sharma, Karine Gonzales, Yanjie Chao, Zaid A. Pirzada, Maria R. Eckert, Jörg Vogel & Emmanuelle Charpentier

Nature 471, 602–607, 31 March 2011.
doi:10.1038/nature09886

This week’s superb summary and analysis by Josien van Wolfswinkel:

Over the last couple of decades CRISPR has become known as the prokaryote version of an adaptive immune response against viruses. In contrast to the vertebrate version of immunity, this system functions not on a protein level, but via recognition of the DNA sequence of the invader by crRNAs.

The CRISPR system consists of two units that together form the CRISPR locus: the “clustered, regularly interspaced short palindromic repeats” (CRISPRs), and the CRISPR associated (cas) proteins which are encoded as a group directly adjacent to the CRISPRs. A CRISPR sequence consists of ~30nt long repeats which are interrupted by ~40nt sequences that can be derived from phages. The whole sequence is initially transcribed as a long single-stranded pre-crRNA, from which mature crRNAs consisting of a single spacer sequence with some surrounding repeat sequence are processed by the cas proteins. Based on the combination of proteins encoded in the cluster, 8 types of CRISPR clusters can be distinguished. Most of them contain an enzyme that is known to perform the RNA processing reaction, however in two types of clusters (Nmeni and Dvulg) no processing enzyme had been found so far.

In this paper Delcheva et al. describe a novel pathway for the processing of mature crRNA from the pre-crRNA primary transcript. First, the authors established that many clinical isolates of Streptococcus pyogenes contain two types of CRISPR clusters (Nmeni and Dvulg), of which only the first type is expressed. The S. pyogenes Nmeni cluster produces mature crRNAs with a 5′ monoP, suggesting that these are not primary transcripts, yet none of the known CRISPR processing enzymes is encoded among the cas genes of this cluster. The authors identified a locus adjacent to the CRISPR cluster, which produces two primary transcripts and one processed RNA species at high levels, and named this the trans-activating crRNA (tracrRNA). Closer inspection of the locus sequence revealed a 25 nucleotide stretch present in both primary tracrRNA transcripts that has almost perfect complementarity to part of the repeat in the pre-crRNA primary transcript. Notably, the 5′ end of the processed tracrRNA, as well as the 3′ end of mature crRNAs are located within this basepairing region.

The authors used deletions of both tracrRNA and pre-crRNA loci to show that the production of mature crRNA depends on co-processing with the tracrRNA. The positioning of the cleavage sites on the pre-crRNA/tracrRNA duplex shows a 2nt 3′ overhang, suggestive of processing by an RNase III type enzyme. Indeed deletion of the S. pyogenes RNase III gene rnc abolished the co-processing, and recombinant Rnc was sufficient to drive co-processing of pre-crRNA and tracrRNA in vitro. In vivo however, the cas gene csn1 (but none of the other cas genes) was also required.

The authors then asked whether this CRISPR cluster can effectively confer resistance to invading phages or plasmids. They created a plasmid containing a sequence identical to one of the spacers in the CRISPR locus and found that this plasmid cannot be transfected into wildtype S. pyogenes, but is accepted by mutants in pre-crRNA, tracrRNA, rnc, or csn1.

Finally the authors identified tracrRNA loci in other species carrying the Nmeni type CRISPR cluster, and show that RNA from these loci is expressed and processed with similar dynamics as in S. pyogenes. Therefore, the mechanism described in this paper may well be a general mechanism for processing crRNA from Nmeni type clusters.

Technically, the paper is solid, but it is the conceptual aspects of it that make it remarkable. First, there have been many indications that CRISPR clusters have been transmitted between bacteria by horizontal gene transfer, and so far the clusters seemed to function as autonomous entities, which are independent of the rest of the bacterial genome. The mechanism for crRNA processing described in this paper is the first report of CRISPR dependency on unlinked loci (i.e. the Rnc that is required for the processing is not present in the CRISPR cluster). It is unclear whether this is due to a loss of independence of the Nmeni cluster, or whether the Nmeni cluster actually represents an ancestral minimal version of the CRISPR system. Second, these Nmeni crRNAs lack the 8nt 5′ repeat-derived tag–defined by positioning of the processing enzyme– characteristic of previously studied crRNAs in other species. The presence of these repeat-derived tags has also been shown to be important for the discrimination between self and non-self. In contrast, in the Nmeni-specific mechanism described in this paper, the repeat-derived tag is on the 3′ end, and therefore it is this end of the mature crRNA that is precisely defined (in this case by the Rnc and the base-pairing with the tracrRNA). This suggests that the mechanism of self versus non-self discrimination could be similar even between classes of crRNAs that differ in the positioning of their repeat-derived tags at opposite ends.

Finally, there is the very tempting parallel between this type of crRNA processing in prokaryotes and the diverse regulatory small RNA pathways in eukaryotes. Both systems use the functionality of an RNase III type enzyme to create small RNAs that are used in a silencing response. The remaining parts of the biogenesis pathways and the modes of silencing differ substantially, but nevertheless, it is interesting that the use of RNase III for regulatory small RNA processing is so widespread.

Translational Pausing Ensures Membrane Targeting and Cytoplasmic Splicing of XBP1u mRNA

Kota Yanagitani, Yukio Kimata, Hiroshi Kadokura, Kenji Kohno

Science Vol. 331 no. 6017 pp. 586-589, 4 February 2011.
DOI: 10.1126/science.1197142

This week’s articulate summary and analysis by Anna Drinnenberg:

This paper from Yanagitani et al. further characterizes a mechanism involving an unconventional splicing event of the XBP1 mRNA that controls a cellular response to the accumulation of unfolded proteins in the endoplasmic reticulum (ER). For this splicing event to occur, it is thought that the nascent XBP1u (u – unspliced) protein, while still part of the mRNA-ribosome-nascent chain (R-RNC) complex, recruits the whole complex to the ER membrane, where a protein localized within the membrane processes the XBP1u mRNA into its spliced XBP1s (s – spliced) form. The HR2 region of the XBP1u protein that was suggested to be important for this recruitment, however, is located at the very C-terminus of the protein. Therefore HR2 is exposed from the ribosomal tunnel for only a brief period before translation is finished, which leads to the question of how the R-RNC complex can still persist while being recruited to the ER by HR2.  Therefore, the authors hypothesize that a translational pause must occur to ensure sufficient time for the ER-recruitment of the R-RNC complex and splicing of the XBP1u mRNA.

Using in vitro studies, they convincingly showed a pause during translation of the XBP1u protein by detection of translational intermediates composed of a tRNA covalently attached to the nascent polypeptides, whereas translation of XBP1s protein that had a different C-terminal region lacking HR2 showed no delay. Furthermore, the authors narrowed down the region responsible for the translational pause to the evolutionary conserved C-terminal part of the XBP1u protein, namely the last 26-amino acids. Exchanging many of these amino acids for alanine decreased or abolished translational pausing, whereas mutating a serine residue at position 255 (S255A mutant) increased pausing, interestingly. The authors try to explain the effect of the S255A mutant by hypothesizing that this residue might ensure an appropriate efficiency of translational pausing to recruit the R-RNC complex, while preventing undesired translational arrest (which would not relieve the spliced mRNA). For all subsequent analysis they included two mutant constructs (in addition to the S255A construct) that nearly completely abolish translational pausing.

After showing that translational pausing also happens in vivo, they demonstrated that in vitro it also appeared to be required for efficient membrane recruitment through the HR2 region. An in vivo demonstration of the R-RNC recruitment would still be worthwhile since the complexity of the intracelluar environment through which such an R-RNC complex would have to traverse is certainly much greater than their in vitro system.

Returning to the molecular effects of membrane recruitment of the R-RNC complex, the authors showed that translation pausing is important, but not absolutely necessary, to ensure efficient splicing of the XBP1u mRNA. While there was certainly a decrease in splicing efficiency without pausing, the effect seemed to be relatively small. However, it is still possible that this subtle decrease in splicing efficiency has greater physiological consequences during a response to ER stress. Mutating a combination of the amino acids that contributed to translational pausing, instead of one at a time, might have also yielded bigger effects on splicing.

Overall, the authors performed a very thorough study showing translation pausing of the XBP1u mRNA and demonstrating its importance for splicing of the XBP1u mRNA. The authors speculate that a physical interaction between the nascent peptide and the ribosomal tunnel might explain the translational pause as it has been observed for the bacterial SecM and TnaC proteins. An important follow-up question is: Is translational pausing a more widespread phenomenon than can be predicted based on the amino acid composition of a protein? As was suggested in an accompanying perspective by David Ron and Koreaki Ito in Science, recent data mapping the progression of ribosomes across mRNAs at single nucleotide resolution (Ingolia et al., Science 2009) will be crucial in answering this question.

Global impact of RNA polymerase II elongation inhibition on alternative splicing regulation

Joanna Y. Ip, Dominic Schmidt, Qun Pan, Arun K. Ramani, Andrew G. Fraser, Duncan T. Odom and Benjamin J. Blencowe

Genome Research, Advance Online 16 December 2010.
doi:10.1101/gr.111070.110

This week’s enlightening summary and analysis by Charles Lin:

There has been a growing appreciation in the last decade that RNA processing and transcription do not occur in isolation. Events thought to be exclusively transcriptional, such as chromatin modifications, elongation rate, and elongation complex factors have been found to interact with almost every aspect of RNA processing—from 5’ cap formation to 3’ end processing and export. Moore and Proudfoot have an excellent 2009 review that describes these interactions in detail.

Of focus this week in RNA journal club is the relationship between alternative splicing (AS) and elongation capacity of the RNA Pol II complex. Ip et al., use multiple techniques to reduce the elongation capacity of RNA Pol II and assay the effect on alternative splicing. They use these perturbations of RNA Pol II to examine two longstanding models coupling elongation/splicing interactions.

The first, the kinetic model, reviewed in Kornblihtt 2006, states that the speed of the RNA Pol II can influence AS through competitive kinetics of 3’ Splice Site choice. The second, the recruitment model, most recently reviewed by Luco et al., 2011 emphasizes the role of chromatin adaptor complexes to couple the splicing apparatus to chromatin modifications and the RNA Pol II.

Ip et al., inhibited RNA Pol II elongation through multiple mechanisms. Inhibition caused a majority of genes to decrease mRNA expression. AS was assayed with a custom array platform that interrogated exon inclusion/exclusion. The authors did not find a compelling trend towards inclusion or exclusion. Instead they focused on the set of genes that experienced exon inclusion. These genes were enriched for splicing factors and in some cases exon inclusion resulted in the addition of a premature termination codon leading to NMD mediated down regulation. This presented an enticing mechanism for coupled coordinated regulation of elongation and splicing machinery. When elongation is down regulated, it causes splicing machinery to be consequently down regulated through NMD.

The authors also find that inclusion of exons is often associated with increased RNA Pol II density flanking the exon. Increased RNA Pol II may be a function of polymerase stalling at the exon or simply a result of a slower elongating complex. It’s unclear whether the RNA Pol II accumulation represents an opportunity for a weaker 3’ Splice Site to be recognized (kinetic model) or additional recruitment of adaptor factors.

Ip et al.’s findings do not discredit one model or the other, and indeed it’s possible for these two models to co-exist. One potential reason for this is that the elongation kinetics of RNA Pol II are intrinsically linked to the ability of the elongating complex to recruit elongation factors/chromatin adaptors. In particular, several of the methods employed by Ip et al. to inhibit elongation kinetics do so by reducing or eliminating serine phosphorylation on the RNA Pol II C-terminal domain repeats. Phosphorylation of these repeats is responsible for both enhanced processivity of the enzyme and also serves as a scaffold for many elongation specific factors.

At the end of the day, the authors propose a set of enticing models built upon their observations. That many of these splicing changes were reproduced through orthologous methods lends weight to the idea that globally, splicing and elongation are coupled processes, and regulation of one may lead to coordinated regulation of the other.