Tuesday, July 16, 2013

Expanding the PIWI repertoire

The PIWI module directly binds a small RNA transcript which in turn targets a reverse complementary substrate, a remarkable form of RNA-based regulation in the cell which has been linked to a continually-expanding list of pathways including transcript silencing, splicing, chromatin dynamics, DNA break repair, and viral defense, to name a few. The PIWI module was identified well over a decade ago but, until now, the classical PIWI family found in PIWI and Argonaute proteins has remained the only known family. In a very-recently published paper [http://www.biologydirect.com/content/8/1/13] from our group, we characterize two novel families of PIWI domains, one found in bacteria and the other in eukaryotes. The bacterial version, dubbed the pPIWI_RE family (in part overlapping with what used to be called the domain of unknown function: DUF3893), is predicted to function in a defense system against invasive phages or plasmids while the eukaryotic version, the medPIWI family, is the defining domain of the human Med13 protein and its eukaryotic orthologs which are crucial regulators of the Mediator complex—a complex required for transcriptional initiation of most eukaryotic genes and one of the primary discoveries behind the awarding of the 2006 Nobel Prize for Chemistry [http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2006/advanced-chemistryprize2006.pdf].

Perhaps the overriding question after discovery of these new families was whether they could bind small RNAs to effect function similar to the classical PIWI module. The PIWI module as defined in Pfam [http://pfam.janelia.org/family/piwi] actually consists of two distinct domains: an N-terminal Rossmannoid domain which utilizes a unique constellation of conserved residues to bind the 5’ end of the small RNA and a C-terminal domain belonging to the RNAse H nuclease fold which, while often nuclease-inactive, contributes conserved residues primarily interacting with the 4th and 5th nucleotides measured from the 5’ end of the bound small RNA. Careful comparison of both new families with the classical family revealed conservation of amino acids at positions crucial for small RNA binding. Perhaps most notably, the 5’ end-binding constellation of residues was conserved, indicating the new PIWI modules could bind either processed RNAs with exposed 5’ ends or the 5’ transcribed end of a nascent RNA transcript. (Note: The final PDF version of our paper appears to have been produced with low resolution figures so we recommend that the reader directly download the author-supplied images from the HTML version of the open-access paper).

If these novel PIWI modules are capable of binding small RNAs, what are they binding? After considering several lines of evidence, we hypothesized the bacterial pPIWI_RE domain is likely binding the 5’ RNA end of the RNA component of the R-loops (RNA-DNA hybrids) characteristic of replicating invasive plasmids/phages. The reasoning: first, we were unable to detect any conserved, genomically-encoded small RNA transcripts around the pPIWI_RE-encoding gene or its operonic neighbors. Second, the pPIWI_RE domain is tightly-linked in an operon with the DinG helicase, a helicase which has been shown in distinct contexts to specifically interact with R-loops. Finally, such a target enforces selectivity on a defense system weaponized with a potential lethal Restriction Endonuclease fold endoDNase which appears to lack any other method for distinguishing “self” vs. “non-self”. This situation might be compared with a subset of the “Type U” CRISPR/Cas systems which similarly have a DinG helicase combined with Cas7 and Cas5 clade RAMPs.

The Med13 protein is a crucial component of a subcomplex regulating the Mediator complex. This subcomplex transiently associates with essentially all promoters, but only associates strongly at a promoter following activation of an as-yet undetermined physical switch which enacts a conformation change [http://genesdev.cshlp.org/content/23/4/439]. We postulate that medPIWI binding to a small RNA constitutes this switch, with the most likely source of this small RNA being cis-generated promoter-derived small RNA transcripts. Recent research has indicated that small RNAs are generated from divergent transcription (transcription on the forward and reverse strands) at and around transcriptional start sites (TSSs) [http://www.nature.com/ng/journal/v41/n5/full/ng.312.html , http://www.nature.com/nature/journal/v480/n7377/full/nature10492.html]. The quantity of these small RNAs at any TSS is roughly proportional to the strength of expression of a gene, dovetailing nicely with the observation that the Med13-containing subcomplex associates most strongly with highly-expressed promoters [http://genesdev.cshlp.org/content/23/4/439].

Several questions remain to be answered, but these discoveries potentially open up exciting new avenues of research. The pPIWI_RE module appears to represent the second RNA-dependent restriction system in prokaryotes after the CRISPR system. It could potentially be exploited as a method for cleaving target DNA using an RNA guide as is being exploited in recent studies using certain types of CRISPR systems. The medPIWI module could provide insight into both the mechanism by which Med13 and its allied proteins modulate Mediator transcriptional activation and the function of small RNA generated near promoter regions.