Unmasking the role of maternal transcript UTRs in reproductive fecundity

Project Summary: My lab is interested in defining how the maternal load is established during oogenesis and decoded af- ter fertilization. We know the identity of the most important maternal transcripts and maternally supplied RNA- binding proteins. We know which factors are required for germline development, oocyte maturation, and pat-

tern formation in early embryogenesis. But we do not yet know which regulatory events are most important for reproduction, or what mechanisms coordinate regulation in space and time. And we do not yet know how envi- ronmental factors modulate maternal regulation, or why reproduction is so exquisitely temperature sensitive.

The nematode Caenorhabditis elegans is an exemplary tool for the study of germline development and reproductive biology. The worm is easy to culture, has a short generation time, and a large brood size. The an- imal is transparent making visualization of fluorescent reporters in live animals straightforward. RNAi is highly

efficient in this species— pathways exist to take up double stranded RNA (dsRNA) from the environment and spread it throughout the worm, enabling RNAi by soaking the worms in dsRNA, or by feeding the worms bacte- ria that express dsRNA, a property that has enabled routine genome-wide RNAi screening. Now, thanks to

CRISPR / Cas9, it is possible to make both targeted gene deletions and targeted gene replacements in C. ele- gans a relatively straightforward way. In preliminary data, we used CRISPR / Cas9 genome editing to make 3´UTR deletions in three critical maternally supplied genes: glp-1 (Notch), mex-3 (hMEX), and oma-1 (Tis11). All of these genes are highly

conserved from worms to mammals, where they play key roles in specifying cell fates during early embryogen- esis prior to the onset of zygotic transcription. While the 3´UTRs of glp-1 and mex-3 are necessary and suffi- cient to pattern their expression in the germline, we discovered to our surprise that neither 3´ UTR is required

for reproduction under standard laboratory conditions. By contrast, the oma-1 3´UTR is absolutely essential, but only in the absence of a functional copy of the redundant gene oma-2. Interestingly, under slightly elevated temperature growth, deletion of the mex-3 3´UTR causes a highly penetrant maternal effect lethal phenotype. We further show that the 3´UTR is a negative regulator of mex-3

levels in the germline, but a positive regulator in the embryo. The primary role of the mex-3 3´UTR is to buffer expression under fluctuatating temperature. These data provide the basis for our hypotheses to be tested: 1) fluctuating temperatures effect the abundance and translation of maternal mRNAs, which may be a major

cause of the temperature sensitivity of reproduction observed throughout the animal kingdom, and 2) that post- transcriptional regulation of maternal RNAs is necessary for reproductive robustness during stress as opposed to being an essential requirement for fertility. We will comprehensively test these hypotheses through an inte-

grative, multi-disciplinary approach including molecular genetics, genomics, and mechanistic experiments.