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Uncovering the unexpected: New visions from the 3’ end of squint

Posted by , on 31 July 2012

Here I share the background story on my graduate work that was recently published in Development, “Dorsal activity of maternal squint is mediated by a non-coding function of the RNA”: I first joined Karuna Sampath’s group at Temasek Life Sciences Laboratory in 2005 during my undergraduate days. It was an exciting year in the lab because Aniket Gore, a former student, had published an interesting story on the identification of asymmetrically localized maternal sqt RNA as the earliest dorsal marker in zebrafish. I found the role of sqt in dorsal specification immensely intriguing, therefore I started working on it as a graduate student.

However, very soon, my project was shrouded with controversy.  Colleagues in the field had challenged Aniket’s findings, and I was following up on his work.  The first hurdle I encountered was to convince Karuna (yes, you are reading this correctly) that sqt RNA has a function independent of Sqt protein.  The sqt insertion mutants were thought to be nulls. I was stunned when I saw that sqtcz35 RNA elicited transient dorsal expansion in wild-type embryos. I remember showing Karuna the sqtcz35 RNA-injected embryos stained for goosecoid and chordin, excited that I had identified a non-coding function. But the first thing she asked was, “Are you sure your sqtcz35 prep is not contaminated with traces of wild-type sqt?”  I re-transformed pCS2+sqtcz35, made countless mini-preps, sequenced them, synthesized capped RNA, injected and assayed each for dorsal expansion in the embryos to show there was no contamination.  In any case, wild-type sqt RNA did not show transient dorsal expansion, so I knew this was something unusual.

The series of experiments that followed was rather straight-forward. I tested different forms of non-coding sqt RNA (sqtmut RNAs) and found that even heterologous sequences fused with sqt UTR could induce dorsal, and this does not require Oep-dependent Nodal signaling. Since the dorsal activity was in the sqt UTR, Steve Cohen and Mark Featherstone, who serve on my thesis committee, asked if microRNAs had any part to play. MZdicer embryos were still able to respond to the sqtmut RNAs, ruling out miRNAs in this process.  This was a new function I had found in the sqt 3’UTR.

No words can describe my excitement at being able to share my findings with the readers of Development. Although the journey was quite arduous, I am pleased that my work has addressed some of the issues surrounding the debate regarding maternal sqt and DV axis formation, and that I have found an interesting area of biology that can be further mined upon.  My focus is now to understand the precise mechanism by which maternal non-coding sqt functions in dorsal axis formation.

Shimin Lim

Shimin Lim, Pooja Kumari, Patrick Gilligan, Helen Ngoc Bao Quach, Sinnakaruppan Mathavan, & Karuna Sampath (2012). Dorsal activity of maternal squint is mediated by a non-coding function of the RNA Development, 139 (16) : 10.1242/dev.077081




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