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 4^th and 5^th 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.

On the origins of the bacterial transcription apparatus

Many years ago, a little after the first RNA polymerase structures were solved, we obtained several remarkable insights into the core transcription apparatus of life.  We were the first to show that the RNA polymerase subunits, cognates of the bacterial beta and betaprime subunits, contain recognizable, evolutionarily conserved domains and that each of these subunits contribute a double-psi beta barrel domain to the active site. We also showed that the polymerase subunits accreted several other domains in a lineage-specific manner, which differ between the archaeo-eukaryotic and the bacterial subunits, and even within the bacterial versions. Our study  also established the common origin of the RNA-dependent RNA polymerase involved in RNAi and the cellular DNA-dependent RNA polymerases (Click to access [Reference1] [Reference 2]).

Recently, we conducted a reanalysis of the bacterial transcription apparatus and from this study emerged several new insights that have refined or redefined our thinking on the origins of the transcriptional apparatus [Click to read].  Some of these new findings were discussed at greater length with a leading researcher in the field of transcription and a part of the correspondence is reproduced below as questions and answers.

Question: One of the new points uncovered in this study is the shared evolutionary ancestry of the archaeo-eukaryotic TFIIB and the bacterial sigma factor, based on structural similarity of the cognate HTH domains that interact with similar sites on the archaeo-eukaryotic and bacterial RNAP, respectively. Is the homology in any way reflected on the sequence level?"

Answer: The simple answer to the question is yes -- we can detect using different sequence profile methods statistically significant sequence similarity between the TFIIB and Sigma HTHs. In conclusion there is no doubt about their evolutionary relatedness and descent from a common ancestor (for example a comparisons of the HMMs of archaeal TFIIB orthologs with Sigma70-like superfamily using profile-profile comparisons; e.g. HHpred; gives p=1.8e-6 and probability of 86% and many more such lines of support). .

Question: Could the similarity between the transcription factor-RNAP interactions in the bacterial holo-RNAP and the RNAPII-TFIIB / RNAP-TFB complexes be a case of convergent evolution?

Answer:  Several aspects of the interactions of the bacterial and archaeo-eukaryotic RNAPs are very likely to be convergent and we have no counter-argument in this regard. The main point is the orthology of sigma and TFIIB despite being distantly related (which seems likely now to us).

Question:  The current consensus in the field is that there are no real sigma homologs in archaea, or eukaryotes. It is argued that the LUCA RNAP could have initiated in a transcription factor-independent manner, and that the sigma and TFIIB/TFB-related factors emerged in evolution following the split of the bacterial and archaeo-eukaryotic lineages. 

Answer with a bit of history from LA): Many moons ago in our early days of sequence analysis we had studied the HTHs in considerable depth. One thing that became clear was that all these HTHs, be it sigma or TFIIB certainly shared a common origin (a view articulated in these papers that we wrote several years later pmid: 10556324 [Click to access]and another in 2005 pmid: 15808743 [Click to access]). As a result of these investigations it became clear that TFIIB (of course including TFB)/cyclin/RB and sigma are *real homologs*, but throughout that period the issue remained as to whether they were *real orthologs*. The reason being many other basal HTHs also show significant similarity to each. Of course, we could rule out things like TFIIE wHTH and MBF-like 4-helical HTHs from contending for ortholog-hood with sigma because they belong to different lineages of HTHs that have their own clear-cut bacterial cognates. But sigma remained unclear. In course of the above mentioned papers, I took a stance that indeed sigma and TFIIB, while being genuine homologs, were independent recruitments as basal TFs which interacted with the RNAP. But since 2005 we got an opportunity to understand the RNA polymerase evolution better using the template of our earlier studies on these proteins (12553882 [Click to access], 15194191 [Click to access]) aided by the various versions from diverse selfish elements that offered potential evolutionary intermediates. So in conclusion it became clear that they began as RNAPs that could have initiated transcription factor-independently, especially given that they lacked any specially adaptation to interact with TFs or had inbuilt HTH domains that might have substituted for the TF. But the beta cognates of the RNAPs of cellular life were unified by one striking synapomorphy in the form of the insertion of the SBHM within the catalytic DPBB domain that could not have been convergence. The emergence of this insert would indicate the emergence of interactions of a DNA-bound TF as it plays this role in all the three superkingdomains of life and is absent in the RdRP-like RNA polymerases (e.g. YonO) and RNAPs of selfish elements such as the NCgl1702-type RNAPs. This, taken together with the homology of the sigma and TFIIB, and the fact they have double HTHs, made us reconsider our former position and accept the more simple explanation of sigma and TFIIB being orthologs, albeit distant in sequence. Of course this divergence in sequence is not surprising with lot of independent action happening around them such as emergence of TBP in the archaeo-eukaryotic lineage etc.

Question:  If the primordial ribozyme RNAP evolved into the extant multisubunit RNAP by recruiting a dimeric DPBB protein cofactor which usurped the active site, and over time increased the subunit complexity to result in the extant multisubunit RNAP, where does that leave the single subunit enzymes? Did they emerge later, earlier, or at the same time? Different members of extant single subunit nucleic acid polymerases have all these activities (RNAPs, DNAPs, RT etc.). Assuming that they would have predated multisubunit RNAPs, when did the change of guard occur, and for what functional reasons/selective advantages?

Answer: Currently we can list the following major independent inventions of RNA polymerase activity:
Within the RRM-like fold or the classical palm-containing polymerases: 1.1) The RNA viral RdRPs; 1.2) the THG1 (5'->3')-CRISPR-like RNA polymerases (at least some are RdRPs) and the 1.3) Phage T7-like RNAPs. Within the RRM-like fold with a flange: 2) archaeo-eukaryotic type primases. Within the TOPRIM fold: 3) DNAG-like primases. Within the pol-beta fold: 4) CCA-adding enzyme-poly A polymerase-like. Within the DPBB fold: 5) The double barrel RdRPs and DdRPs.

While there were many inventions of RNA polymerases, the following observations seem to hold: The RNAPs in the group 1.1 are the main replicative enzymes that replicate RNA in independent replicons. While the double-psi barrel RdRPs replicate small RNAs in the eukaryotic RNAi system, there is no evidence currently for them being dominant replicative enzymes of large replicons. The RdRPs in group 1.1 are further closely related to the replicative reverse transcriptases, which appear to have a single origin. On the other hand, representatives from 1.1, 1.3, 2, 3 and 5 can be associated with replication in the context of the synthesis of the RNA primer for DNA replication. Additionally, the primpols from group 2 can replicate DNA after initiating it with a RNA polymerase activity for priming. We are of the opinion that indeed RNA was more likely the primary nucleic acid (supported by: 1) its catalytic and replicative capacity; 2) its association with polypeptide templating, and 3) the priming problem making DNA a difficult starting genome. This conclusion, combined with the above observations regarding the RNAPs of group 1 and the relationship to RTs, leads us to propose that the polymerases from the 1.1. group were the first to emerge. They enabled the rise of DNA genomes with the origin reverse transcribing ability as they radiated. The emergence of DNA in turn offered a new niche for RNA polymerases due to the priming problem. This selective force appears to resulted in the emergence of multiple RNA primer synthesizing enzymes (early representatives of 1.3, 2, 3 and 5) as evidenced by the above observations. Even at this stage it is possible that there was a reverse transcribing intermediate in replication, which also helped solve the transcription problem for DNA replicons. The rise of large DNA replicons appears to have placed the pressure for transcription-specific RNAPs. This unique niche appears to have favored two major groups of RNA polymerases -- 1.3 and 5, but in the lineage leading to the cellular replicons 5 seems to have dominated. We suspect that the elements of the architecture of the double-psi beta barrel polymerases allowed them to be more effective transcription enzymes due to: 1) their ability to initiate transcription at internal sites independently of a replication origin signal for which the other enzyme were optimized; 2) their offering interfaces for regulation -- in particular the distinctive bihelical extension preceded by two extended segments forming a standalone haripin in beta-prime. The latest analysis of the evolution of double-psi beta barrel RNAPs suggests that they two began as a fusion of two DPBBs in a single polypeptide followed by a split prior to LUCA.

Question: Could these accretions have been responsible for improved regulatory potential or higher fidelity? In that context it is noteworthy than no single subunit RNAP can 'backtrack' and undergo transcript cleavage.

Answer: The addition of subunits, basal TFs and SBHMs and other domains do clearly point in the direction of continuous evolution favoring higher fidelity and regulatory potential. In particular it might have helped provide robustness to this central cellular system in face of mutational "attack" -- over-engineering.The last point of the question is of note and might have been a selective force in the later evolution of the RNAPs.


Question: Since the RNAP are predicted to have their origins as ribozymes and went through an RNA-protein stage, why is the ribosome apparently slower in losing its RNA components, as compared to nucleic acid polymerases.

Answer: First, regarding the ribosome where the RNA plays a role in peptidyltransfer: We have recently extensively studied the emergence of peptide bond forming activity in protein enzymes (pmid: 20023723 [Click to access], 20678224 {Click to access]). There were at least 11 independent inventions of peptide ligase activity, but an examination of each of these suggest that they are unable to handle the reaction in an amino acid independent manner. This inability of the protein peptide ligases might have allowed the RNA to persist. Further, a look at the other ancient ribozyme RNAse P suggests that shape selective recognition of nucleic acid structures, which is a feature it shares with the ribosomal RNAs might be a key factor that cannot be entirely reproduced by proteins. In these cases the ribozymes certainly would persist. Further RNA is also a better scaffold than proteins in certain contexts and it continues to be used as such in contexts like the eukaryotic Polycomb RNAs and HOTAIR. So, we do not see a need for RNA to be displaced in every case. Our original ribozyme displacement hypothesis was based on the observations like: 1) Several of the ancient enzymes are homologs of non-enzymatic ancient domains that bind RNA and 2) In cases like RNAseP, the protein component increases the catalytic rate of the ribozyme by potentially increasing local affinity metal ion. This offers a pre-adaptation for the protein acquiring metal-binding dependent catalysis. Now, given the new information on the evolution of the doublepsi beta barrel RNAPs, it appears that the RdRP activity might be a secondary innovation. Hence, it is conceivable the DPBB domains were merely nucleic acid binding cofactors in an already protein dominant world and its associated nucleic acid might not have had any catalytic activity. It is becoming increasingly likely that a RNA only world was probably never there (i.e. independent of proteins) and early RNAs at best had restricted catalytic capabilities in the RNA world. It is even possible that right from the beginning the basic reciprocal catalytic cycle involved early RNAs catalyzing peptide-bond formation and protein synthesis (precursor of the ribosome) and the proteins in turn catalyzing the formation of the phosphodiester bond and RNA synthesis.

Question: What about the evolutionary origins of the TBP fold, and of TBP itself? The single fold itself can be found in RNaseHIII and DNA glycosylases but it has not been demonstrated to mediate any direct interactions with DNA or DNA, that emerged later, with TBP in the archaeo-eukaryotic lineage. What happened before that, did the LUCA RNAP initiate TFIIB-sigma dependent? 

Answer: TBP belongs to the larger helix-grip fold (pmid: 11276083 [Click to access]) that includes proteins with various binding capabilities. When we first showed the relationship between TBP and the RNAseHIII N-terminal domain in 2001 (pmid:11582786 [Click to access]), it was the closest to TBP within the helix grip fold. However, since then we found another member of the fold, CCTBP that is as related as the one in RNAseHIII to TBP (PMID: 19089947). Both these are much closer to TBP than the version in the DNA glycosylases. Hence, the evolution of TBP is to be understood in the context of these related domains.  Of these the CCTBP is involved in sulfotransfer along with ubiquitin like proteins. The evidence does suggest that the RNAseHIII TBP domain might interact with DNA-RNA hybrid molecules. Hence, it appears that during the radiation of the TBP family it acquired very distinct activities, but the one associated with primer degradation or RNA-based DNA restriction is a more likely candidate for precursor of TBP the basal TF than CCTBP, which is associated with distinct metabolic activities. However, this might change if a nucleic acid binding activity is demonstrated for the CCTBP domain.

A 40 year old mystery: The identity of the wybutosine hydroxylase and other questions

Figure: Occurrence of Y-base (wybutosine) in the literature using Google Ngram

Modified bases are particularly prevalent at position 37 of tRNA. Being adjacent to the anticodon, these modifications stabilize mRNA–tRNA pairing and assist maintenance of the reading frame during translation. One such complex modified base found at this position in eukaroytic phenylalanine tRNA synthetase is wybutosine (also called Y-base or yW). Over the past 40 years various studies identified the intermediates and enzymes involved in its biosynthesis. Additionally, it was shown that precursors of this modification pathway are present in archaea, suggesting an archaeal origin for this modification.

Although wybutosine is detected in diverse eukaryotes, this base position in tRNA^Phe shows considerable variation. For example, tRNA^Phe in yeast contains wyosine in the same position whereas flies only have 1-methylguanosine. This type of variation can be attributed to gene loss, given that 1-methylguanosine and wyosine are precursors in the wybutosine biosynthesis pathway. This is also supported by the phyletic distribution of the enzymes of this biosynthesis pathway in these organisms. In contrast, mammalian liver extracts, and Geotrichum were shown to contain a further modification; hydroxy/hydroperoxywybutosine, suggesting the presence of a distinct enzyme that catalyzes this step. Until recently, the identity of this hydroxylase was not known.

Using a combination of sequence and contextual analysis, we identified the enzymatic domain involved the biosynthesis of hydroxy/hydroperoxywybutosine. The domain is often fused to enzymes involved in the biosynthesis ofwybutosine precursors and also occurs as a stand-alone domain in metazoans (e.g. C2orf60 in humans). What is remarkable is that it turned out to be a member of the JOR(jumonji-related)/JmjC superfamily. Members of this superfamily are normally characterized as hydroxylases of proteins or histone demethylases. This is the first example of an RNA substrate for a member this superfamily. A few months after our publication, this prediction was experimentally confirmed. The JOR/JmjC belongs to a lineage of protein called the 2-oxoglutarate Fe (II) dependent dioxygenases or 2OGFeDO. In turn, the core of this lineage belong to the double stranded beta helix (DSBH) fold.

This discovery actually unraveled more questions--

Q1. Are RNA substrates ancestral to the JOR/JmjC superfamily or were they derived only in the wybutosine hydroxylase family?

Answer: The use of an RNA substrate as in the wybutosine hydroxylase appears to be a derived condition in this superfamily of proteins.

Q2. What are the inter-relationships between the various JOR/JmjC families?

Answer: All studies until now only used eukaryotic members for phylogenetic reconstruction of evolutionary relationships between various JOR/JmjC families. Using a comprehensive sequence, structure and phylogenetic based approach that included bacterial sequences, we show that the eukaryotes contain 17 major lineages of JOR/JmjC proteins, that were in turn acquired on three distinct occasions from bacteria. Thus, the major groups of JOR/JmjC appear to have diversified in bacteria followed by a transfer of at least one member from each of the three clades to the eukaryotes, prior to the divergence of the heterolobosean-kinetoplastid clade and the remaining eukaryotes. The three major clades are named the histone demethylase-like, FIH1/yW-hydroxylase-like and the MINA/No66-like clade respectively.

In addressing this, we went a few steps further and were able to classify the entire double-stranded beta-helix fold. Here's an interactive site where you can play with our classification. This led to some interesting hypotheses about their evolution.

Q3. What were the roles of the bacterial ancestors of the eukaryotic JOR/JmjC?

Answer: Quite consistently, we observe that bacterial representatives of this superfamily are coded by gene clusters involved in biosynthesis of secondary metabolites, such as pyoverdine-like siderophores and peptide antibiotics. These gene clusters often encode multiple functionally linked dioxygenases, tryptophan halogenase-like oxidoreductases and other enzymes involved in non-ribosomal peptide biosynthesis and modification. Actually, some of these contexts are quite remarkable. For example, in the Synechococcus phage Syn9 one of these gene clusters encodes 10 tandem dioxygenases including the MINA/No66 homolog, Syn9-gp49. The remaining nine dioxygenases belong to the classical 2OGFeDO superfamily. Analysis of these nine dioxygenases suggests that they are all not closely related. They belong to at least five distinct families, including one distinguished by a fusion to tetratricopeptide repeats.

Q4. Are there any other distinct substrates predicted for the eukaryotic JOR/JmjC superfamily?

Answer: Did you know that there are members of the JOR/JmjC family that are membrane associated or secreted? In each of the 3 major clades in eukaryotes, we detected secreted or membrane associated proteins with JOR/JmjC domains. Some members of the FIH1 clade are fused to sulfotransferases. A distinct lineage-specific expansion of MINA/No66 like JOR/JmjC in Monosiga comprises of receptor-like proteins with extracellular JOR/JmjC domains. All of these proteins combine a JOR/JmjC domain with one or more of several extracellular domains such as cysteine-rich GCC2/3 repeats, immunoglobulin, disintegrin or SUSHI domains and with intracellular SH2 or tyrosine kinase domains. These extracellular proteins appear have been recruited for modifying cell surface proteins, probably as hydroxylases similar to leprecan and the prolyl hydroxylase. Further, the receptor-like proteins in Monosiga could also function as sensors of redox conditions that signal via intracellular tyrosine phosphorylation pathways.

Finally, a common misconception is that the N-terminal region of the JOR/JmjC domain, called JmjN in the literature is a distinct domain. Structural analysis shows this to be conserved in all members of the JOR/JmjC superfamily. Further the domain has no independent existence and merely represents a structural extension of the DSBH fold.

Wait, there is much more.... You can read about it here in detail. Feel free to browse the comprehensive supplement.

SZY-20: A centrosomal protein with RNA binding function

Microtubules are organized by the centrosome, a dynamic organelle that exhibits changes in both size and number during the cell cycle. The maintenance of appropriate centrosome size is critical for proper cell division and partitioning of biomolecules and organelles between the daughter cells. However the exact mechanism by which this process is regulated is unclear.

In a collaborative study with Dr. Kevin O'Connell of the NIDDK, we showed that SZY-20, a predicted RNA-binding protein, plays a critical role in limiting centrosome size in the nematode worm C. elegans. Homologs of SZY-20 are present throughout eukaryotes pointing to conserved role for this protein. SZY-20 localizes in part to centrosomes and in its absence centrosomes possess increased levels of centriolar and pericentriolar components including gamma-tubulin and the centriole duplication factors ZYG-1 and SPD-2. These enlarged centrosomes possess normal centrioles, nucleate more microtubules, and fail to properly direct a number of microtubule-dependent processes. Depletion of ZYG-1 restores normal centrosome size and function to szy-20 mutants, whereas loss of szy-20 suppresses the centrosome duplication defects in both zyg-1 and spd-2 mutants. Our results thus describe a pathway that determines centrosome size and implicate centriole duplication factors in this process. Computational analysis showed that SZY-20 contains a two novel protein domains the SUZ and SUZ-C domain which are predicted to be respectively critical for RNA-binding and targeting of ribonucleoprotein complexes. It was also shown to be a part of a large complex of RNA-binding proteins. Mutagenesis of conserved residues in these domains result in loss of SZY-20 function and loss of ability for form RNA-protein complexes. The presence of a RNA-binding domain in SZY-20, a centrosomal protein, suggests that it might be key for partitioning of RNA during cell division.
For more details, click here.

RAGNYA : a novel fold found in functionally diverse nucleic acid, nucleotide & peptide-binding proteins

One of our principal research objectives is to derive a natural classification of the protein universe by unifying diverse protein superfamilies. However, the detection of relationships between these superfamilies is often non-trivial due to extensive divergence or variations, like circular permutations, in their structural scaffolds. This is particularly prevalent in numerous small folds involved in binding of nucleic-acids/nucleotides. One such alpha+beta fold that we recently identified was the RAGNYA fold that includes a diverse group of proteins principally involved in nucleic acid, nucleotide or peptide interactions. Members of the fold include the Ribosomal proteins L3 and L1, the GYF domain, DNA-recombination proteins of the NinB family from caudate bacteriophages, the C-terminal DNA-interacting domain of the Y-family DNA polymerases, the uncharacterized enzyme AMMECR1, the siRNA silencing repressor of tombusviruses, tRNA Wybutosine biosynthesis enzyme Tyw3p, DNA/RNA ligases and related nucleotidyltransferases and the Enhancer of rudimentary proteins. This fold exhibits three distinct circularly permuted versions and is composed of an internal repeat of a unit with two-strands and a helix. We show that despite considerable structural diversity in the fold, its representatives show a common mode of nucleic acid or nucleotide interaction via the exposed face of the sheet.
Click here to read the paper

The NYN domains: novel predicted RNAses with a PIN domain-like fold

We have shown that the Mut-7C module contains a PIN domain RNAse combined with a Zinc ribbon. Thus Mut-7 is a “double-headed” nuclease with a PIN and a 3’->5’ nuclease domain fused together.
Click here to read the paper

Insights into chronic HCV treatment with PEG-IFN-alpha and ribavirin

With Mani Subramanian and Vijay Balan’s groups we studied the global transcriptional profile during the first 4 weeks of treatment of human chronic hepatitis C patients with pegylated interferon alfa (PEG-IFN-alpha). Novel transcription factors potentially involved in secondary gene regulation cascades, a potential dsRNA receptor with a RNA helicase domain related to that found in the HELICARD protein and members of the ubiquitin signaling pathways, including a novel predicted deubiquitinating peptidase were all identified as being up-regulated upon treatment with IFN. This predicted peptidase is a highly derived version of the APG4 family of papain-like peptidases and contains a catalytic histidine that is in entirely different location from that found in the regular APG4-like proteins. The overall findings provide new light on possible physiological effects of IFN-alpha, new downstream signaling pathways and open lines of investigations on the mode of action of PEG-IFN-alpha combination therapy.
Click here to read the paper