The slicing activity of Argonaute proteins, which forms the basis for RNA interference, is deeply conserved, existing in organisms ranging from archaea to humans. Surprisingly, there are still only few demonstrations of conserved usages for Argonaute mediated slicing that help to explain endogenous biology. This is an important question that still remains to be fully addressed, as it would help to explain why RNAi has been so broadly conserved. I’ve addressed this question by making use of various methodologies to uncover unique requirements for the biogenesis and targeting function of select small RNAs necessary for mammalian development.
In the first section, I describe the discovery and characterization of a novel conserved mammalian locus (mir-486) which requires Argonaute2 (Ago2)-mediated slicing for its maturation and function. In the second section, I explain efforts to understand the genome wide small RNA targets of Argonaute2 slicing, and the loading profile of this slicer protein versus non-slicing Argonaute1, via generation of AGO-IP (Argonaute-immunoprecipitation) small RNA libraries. Through these studies, I was able to find a specific signature of Ago2-slicing affecting a unique set of two erythroid miRNAs.
In the third section, I describe the generation and characterization of mouse models directed toward understanding the in vivo requirements for slicing in development. This involves validation of miR-486 as a target of slicing in mouse tissues and the study of developmental phenotypes associated with loss of this slicing activity. I was able to find that Ago2 slicing has a specific role in the erythroid compartment of the hematopoietic system, where it directs the proper maturation and homeostasis of red blood cells. By probing for expression patterns of the effector proteins and their target RNAs, we were able to uncover a co-evolutionary signature between AGO2 and miR-486 that helps to explain the evolutionary rationale for mammalian conservation of slicing.
In summary, the work uncovers an important target of slicing that has co-evolved with a unique signature of Argonaute expression to allow for the proper development of the hematopoietic system. More broadly, this work helps to explain the biological rationale for the long-standing observation of the conserved slicing activity of Argonaute2 that has been retained in mammalian species.
|Commitee:||Schwer, Beate, Whitehouse, Iestyn, Kharas, Michael|
|School:||Weill Medical College of Cornell University|
|School Location:||United States -- New York|
|Source:||DAI-B 81/2(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Biology, Biochemistry|
|Keywords:||Argonaute2, Hematopoiesis, microRNA, miRNA, RNA, Slicing|
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