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Publication Date

2011-04-18

Availability

UM campus only

Embargo Period

2012-04-17

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biochemistry and Molecular Biology (Medicine)

Date of Defense

2011-04-07

First Committee Member

Mary Lou King

Second Committee Member

Feng Gong

Third Committee Member

Murray P. Deutscher

Fourth Committee Member

Richard L. Rotundo

Fifth Committee Member

Isaac Skromne

Abstract

Early embryonic development proceeds without zygotic transcription. Genetic control is executed by maternally inherited mRNAs that are expressed in a temporally regulated manner. To set up the body plan, it is pivotal to both exert translational regulation of maternal mRNAs and to integrate maternal signals that drive cell fate determination. Xenopus nanos1, dead end (dnd) and VegT are maternal messages critical for the germline and somatic development. nanos1 and dnd localize to the germ plasm at the vegetal cortex of oocytes, while VegT occupies a cortical region overlapping with, but broader than, that of the germ plasm. In this dissertation, we observed that unlike other mRNAs, synthetic nanos1 RNA translates very poorly if at all after injection into Xenopus oocytes. We find that a RNA secondary structural element immediately downstream of the AUG start site is both necessary and sufficient to prevent ribosome scanning in the absence of a repressor. nanos1 is translated shortly after fertilization, pointing to the existence of a developmentally regulated activator. These observations unravel a new mode of nanos1 regulation at the post-transcriptional level for eukaryotes that is essential for normal development. Further studies showed that co-injection of dnd and nanos1 into oocytes resulted in nanos1 activation. Consistent with Dnd being the activator, Nanos1 expression was attenuated in the absence of dnd activity. Recombinant Dnd interacted directly with nanos1 RNA in vitro and possessed ATPase activity. Our findings suggested that Dnd is the potential activator for nanos1 translation by directly disrupting the repressive structural element. We previously showed that VegT, the maternal transcription factor, is normally inherited by the primordial germ cells (PGCs). VegT is the endoderm determinant and must be silenced in PGCs to preserve the germline. We showed that a classic Pumilio Binding Element (PBE) within the VegT 3’UTR mediated translational repression of a fluorescent reporter in the germline. Accordingly, a direct interaction between Xenopus Pumilio1 RNA binding domain and the VegT PBE was demonstrated in a band shift assay. The Pumilio protein belongs to the Pum-FBF family that functions in translational repression. We show that Pumilio represses VegT in vivo. Our results suggest that the germline stays unresponsive to the somatic determinant VegT in part by Pumilio- and Nanos-mediated translational repression.

Keywords

nanos; Xenopus; germline; translation

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