Issue 2

RGCB Blog

10/09/2015

Meet the Expert INSIDE T HIS ISSUE:

Post Translational Modification Rakesh S Laishram PhD Gist of the work Significance of Star-PAP phosphorylation Impacts of the study results Challenges and Hurdles The Team Reference

Post Translational Modification For any living cell, extracellular signals that regulate gene expression are fundamental to development, adaptation to the environment and homeostasis. A wellorchestrated cascade of regulation of specific gene expressions controls cellular function and diseases. Knowledge of the molecular mechanism of these regulatory pathways has elucidated the mystery of various human diseases and pathogenesis. Expression of a gene to a functional protein consists of several wellcoordinated processes that begin with remodeling of the chromatin, followed by mRNA synthesis, and translation to specific proteins. Most proteins in humans undergo many post- translational modifications that affect the activity or function of the protein. Protein

Dr. Rakesh S Laishram PhD phosphorylation is one of the most common posttranslational modifications that play important role in intracellular signal transduction. It affects overall properties of the protein and regulates cellular processes such as metabolism, growth, division, differentiation, organelle trafficking, and membrane transport in the cell. Protein phosphorylation is also

common among transcription factors and RNA processing enzymes. Let us hear from RGCB scientist Rakesh S Laishram PhD talk about his work on Phosphorylation regulates the Star-PAPPIPKI interaction and directs specificity toward mRNA targets that was published in the July issue of Nucleic Acids Research.

Rakesh S Laishram PhD Rakesh S Laishram holds a doctoral degree from Centre for DNA Fingerprinting and Diagnostics, Hyderabad. He joined RGCB in July 2012 after gaining more than four years of postdoctoral experience from the School of Medicine and Public Health of the University of

Wisconsin in Madison, USA. This illustrious young scientist has earned many esteemed fellowships such as Innovative Young Biotechnologist Award (IYBA), DBT India, Wellcome Trust, UK-DBT India Alliance Intermediate Fellowship, American Heart

Association Scientist Development Award/Grant and DBT-Ramalingaswami Fellowship. He runs the PTM Research Laboratory in RGCB with his team of graduate students and research fellows.

Meet the Expert

Page 2

Gist of the work. Phosphorylation and target gene expression: “We showed that phosphorylation of Star-PAP is one of the mechanisms by which Star-PAP regulates specific mRNA targets downstream of different signaling pathways. We identified an in vivo phosphorylation site on Star-PAP N-Terminal RNA binding domain (serine 6, S6) that regulates select StarPAP target genes mostly involved in oxidative stress response. This phosphorylation, however, is independent of other genes such as DNA damage signal induced apoptotic gene expression. Strikingly, S6 phosphorylation functions with PIPKIα, and controls overlapping mRNA targets. This, to our knowledge, is the first example of a poly(A) polymerase (or even a polymerase) where specificity of target gene expression is determined by phosphorylation status of the polymerase.”

The study was based on our hypothesis that StarPAP is phosphorylated downstream of signaling pathways that control distinct sets of genes.We reported an in vivo StarPAP phosphorylated residue (serine 6) in the ZF region at the N-terminus. The phospho-deficient (serine 6 to alanine, S6A) but not phosphomimetic (Serine 6 to glutamate, S6E) mutation of Star-PAP resulted in reduced expression and 3′-end formation of target HO-1 or NQO-1 mRNA. S6A

mutation also inhibited StarPAP mRNA binding and interaction with PIPKIα. We characterized the S6 phosphorylation and showed that CKIα phosphorylates the S6 residue on Star-PAP in vivo. S6-phosphorylated Star-PAP is largely nuclear speckle localized and this phosphorylation regulates Star-PAP RNA association with specific pre-mRNAs (HO-1 and NQO-1). Unlike other CKIα phosphorylation site(s) at the PRR on StarPAP, S6 phosphorylation is independent of oxidative

stress signal. S6phosphorylated Star-PAP shares target mRNAs with those regulated by PIPKIα. We demonstrate that S6 phosphorylation regulates the Star-PAP PIPKIα interaction that in turn is required for specific mRNA expression. Our results indicate a mechanism where Star-PAP mRNA specificity is mediated through specific phosphorylation that controls association with the coactivator PIPKIα.

Significance of Star-PAP phosphorylation. Here we show a novel role of phosphorylation on determination of target gene specificity of the poly(A) polymerase, StarPAP. Several functions of protein phosphorylation are established that include but not limited to protein or polymerase activation, nuclear import, DNA/RNA binding, or overall functional change. However, there is no report so far of phosphorylation(s) that control specific gene selection by a polymerase. Moreover, our study also establishes phosphorylation as a key mechanism by which the two poly(A)

polymerases (Star-PAP vs PAPα) have exclusive mRNA targets independent of each other. One of our interesting findings is that S6-phopshorylation regulated genes are also the overlapping targets of key nuclear phosphoinositide signaling enzyme, PIPKIα. This indicates that nuclear phosphoinositide signal functions with S6phopshorylation on StarPAP in the nucleus. We have also demonstrated that S6-phosphorylation is involved in multiple aspects of Star-PAP function – from mRNA binding to

association with signaling molecule PIPKIα. This suggests an important role of phosphorylation in overall regulation of StarPAP function. This S6-

phopshorylation is the first in vivo phosphorylation site identified on Star-PAP and regulate target genes independent of other kinases that phosphorylate elsewhere on Star-PAP. This implies existence of a different signaling pathway that can induce phosphorylation of distinct residues on StarPAP regulating specific transcripts.

Impacts of the study results. More than 60% of human genes have multiple polyadenyation sites at the 3’-UTR. We have shown involvement of Star-PAP in APA site selection of subset of mRNA targets. We demonstrated role of StarPAP phosphorylation as a key determinant of target gene/UTR specificity. Therefore, our study paves a way for understanding the mechanism of APA

regulation of key human genes. Moreover, 3’-UTR regulation is emerging as a critical mechanism of regulation in several diseases. Star-PAP is a key player in this regulation. Thus, defining phosphorylation sites on Star-PAP mediated by CKIα/PKC or other putative kinases on Star-PAP will be key to elucidate how StarPAP can regulate multiple

genes differentially responsible for distinct functions or diseases. Our study also identified a new mechanism where PI4,5P2 signaling is linked with phosphorylation sites on Star-PAP suggesting association of PI signaling in the 3’-end processing complex with kinases such as CKIα.

Page 3

Meet the Expert

Challenges and Hurdles. The major scientific difficulty was to define the phosphorylation site on Star-PAP. I failed to pick the right peptide by Mass Spectrometry sequencing available in RGCB.I was quite fortunate to use the high-end UW Mass Spectrometry sequencing facility during my Wellcome-Trust overseas visit to purify the protein and identify the phosphorylation site However, during revision, we had no other way to use the facility again to address certain questions and thus had to use difficult and

imperative approaches (and sometimes lengthy) to convince the reviewers. We faced the most difficult situation during the cell biology experiments where our experimental volume was large but the access to imaging facility was limited. With our own microscope now, we hope not to face these difficulties in future. We had many technical difficulties as ours was a recently established lab. We had to go through multiple hits and trials as none of the techniques were standardized in the

lab. With a team practically new to these techniques, I had to train them rigorously towards better results. This was literally double the effort as that of a veteran in the field. Relying on classical approaches with the available facilities to answer difficult questions was very challenging too. The administrative hurdles in the process of indenting and receiving consumables took much of our valuable time, but I understand it is unavoidable with the current scientific system in India.

The Team. Amidst all these hurdles, the only solace I had was my team of young researchers. They dedicated long hours without break for tackling this challenge. I am very proud of my very industrious team. It was an amazing experience for all of us working as a team for the first publication of the

lab. Nimmy Mohan, who took the main responsibility for the experiments, characterized the phosphorylation site, defined the kinase that phosphorylates it and performed most experiments for gene regulation. Sudheesh, AP, who equally contributed, did all the cell biology and

imaging experiments. He showed localization of phosphorylated Star-PAP and its cellular properties. He did all the in vivo RNA binding experiments and nearly a dozen of site directed mutagenesis. Nimmy Francis contributed on in vitro RNA binding experiments and kinase assays.

Ref : Phosphorylation regulates the Star-PAP-PIPKI interaction and directs specificity toward mRNA targets

The spirit hold on to.

you

“That is undoubtedly committed teamwork towards a goal. The experiment design, execution and writing the manuscript were definitely teamwork. I have very high aspirations about my team in the future.”