Molecular and genetic analysis of novel Slicer-dependent miRNA pathways in blood Most conserved microRNAs (miRNAs) are generated by a biogenesis pathway that deposits them into an Argonaute effector, guiding them to broad regulatory target networks. Amongst the cohort of four mammalian Argonautes, only Ago2 has catalytic ability to cleave transcripts, an enzymatic activity known as Slicing that underlies experimental RNA interference. Nevertheless, the endogenous biological usage of mammalian Slicing remains largely mysterious. Our previous and ongoing studies provide the unexpected perspective of multiple Slicing-dependent biogenesis strategies that generate both Dicer-independent and Dicer-dependent erythroid miRNAs. These data strongly support our hypothesis that a dominant usage of Ago2 catalysis is to generate specific conserved miRNAs in the blood system. Our extensive preliminary data are the basis of (1) a series of biochemical and genomic experiments to elucidate a novel Slicing-dependent miRNA biogenesis mechanism, (2) genetic studies of novel knockout animals of erythroid, Slicing-dependent miRNAs in normal development, blood homeostasis and leukemia, and (3) molecular genetic analyses that seek to connect dysregulated processes in Ago2-catalytically defective blood system to specific Slicing- dependent miRNAs. These studies will bring new insights on post-transcriptional control of erythroid development, homeostasis, and blood cancer, as well as pinpoint the functional basis of mammalian RNAi to the generation of erythroid-specific miRNAs.

Public Health Relevance

microRNAs (miRNAs) are ~22 nucleotide RNA molecules that guide Argonaute (Ago) effector complexes to target transcripts for repression. Collectively, miRNAs mediate broad gene regulatory networks with substantial impact on disease and cancer. Special Ago proteins, such as mammalian Ago2, have the ability to cleave target transcripts. This enzymatic activity, referred to as 'Slicing', underlies the powerful experimental tecnique of RNA interference, but the endogenous biological significance of Slicing is poorly understood. Our previous and ongoing studies demonstrate that in mammals, Slicing activity is intimately and fundamentally tied to the erythroid system. In particular, we show that there are multiple non-canonical biogenesis mechanisms for erythroid-restricted miRNAs that uniquely require Ago2-Slicing activity. We propose a combination of biochemical, molecular, genetic, and genomewide studies to elucidate the biological requirements of non-canonical erythroid miRNAs, and how they surprisingly underlie the blood defects of Slicing-defective mammals.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL135564-01
Application #
9219500
Study Section
Molecular and Cellular Hematology Study Section (MCH)
Program Officer
Klauzinska, Malgorzata
Project Start
2017-04-10
Project End
2021-01-31
Budget Start
2017-04-10
Budget End
2018-01-31
Support Year
1
Fiscal Year
2017
Total Cost
$698,647
Indirect Cost
$291,035
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
Research Institutes
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065