The laboratory research focus is to understand the pathways of how small regulatory RNAs are generated, how they exert their gene regulatory function, their role in the self-renewal and pluripotency of embryonic stem (ES) cells, and their relevance to human disease. RNA interference (RNAi) describes the recently identified phenomenon whereby small non-coding RNAs can silence gene expression. It is emerging that cells possess a wide repertoire of tiny regulatory RNAs that are critical for a variety of biological pathways and can repress genes via numerous mechanisms. For posttranscriptional gene silencing, microRNAs (miRNAs), and small inhibitory RNAs (siRNAs), function as guide molecules inducing mRNA degradation or translational repression. In mammals, hundreds of miRNAs have been identified, and have been implicated in controlling diverse developmental pathways. Indeed, recent predictions indicate that over one third of all human genes are targeted by miRNAs.
miRNA-processing in stem cells and cancer
Recent work has provided much insight into the molecular machinery of miRNA biogenesis. MiRNAs are generated by a two-step processing pathway that begins in the cell nucleus where long primary miRNA transcripts (pri-miRNAs) are specifically cleaved by the ‘Microprocessor’ complex to yield ~60-70 nt precursor miRNAs (pre-miRNAs). Biochemical studies identified that the Microprocessor complex comprises the RNAse III Drosha, and its essential cofactor DGCR8. The pre-miRNAs are exported into the cytoplasm where they are processed by Dicer to ~22 nt miRNAs. One strand of the mature miRNA is incorporated into the RNA-induced silencing complex (RISC) that recognizes target messenger RNAs (mRNAs) based on sequence complementarity between the guide miRNA and the mRNA transcript and results in either Ago2-mediated endonucleolytic mRNA cleavage or translational repression.
Perturbation of the miRNA biogenesis pathway impacts the differentiation potential of mouse embryonic stem cells. Moreover, a selective block in miRNA processing has recently been observed in embryonic stem (ES) cells, embryonal carcinoma (EC) cells, and primary tumors. These data support the notion that miRNAs are important for cell differentiation and that unidentified mechanisms exist in stem cells and certain cancers to prevent miRNA-mediated cell differentiation. Using biochemical and molecular biological approaches we aim to identify the mechanism of this miRNA-processing block in stem cells and cancer cells and also to identify particular miRNAs that mediate ES cell differentiation.
Role of miRNAs in stem cell self-renewal and differentiation
It is emerging that miRNAs are important mediators of cell fate decisions and have been implicated in numerous developmental pathways. Until recently, it was thought that miRNAs are required for stem cell viability. This was based on the phenotypes observed by genetic ablation of Dicer in mouse embryonic stem (ES) cells and the identification of a small number of miRNAs that are enriched in cultured ES cells. However, effects of Dicer deficiency are not necessarily attributable to miRNAs as Dicer is also responsible for generating other classes of small RNAs including small inhibitory RNAs (siRNAs). Importantly, recent genetic evidence suggests that miRNAs may be dispensable for stem cell self-renewal. DGCR8, a component of the Microprocessor complex is essential for processing pri-miRNAs. Moreover, unlike Dicer, its function seems to be dedicated to the miRNA biogenesis pathway. DGCR8 knockout ES cells are viable despite the absence of miRNAs. Significantly, DGCR8-/- ES cells are unable to differentiate, suggesting that miRNAs are not required for stem cell self-renewal, but rather are required to elicit cell differentiation.
In collaboration with the Harvard Stem Cell Institute Therapeutic Screening Facility, we are performing a high throughput chemical screen to identify specific inhibitors of the Microprocessor complex. We hypothesize that these chemicals, by mimicking the effects observed in DGCR8 knockout stem cells, will enable us to expand stem cell populations by preventing miRNA-mediated cell differentiation. We predict that chemical inhibition of miRNA biogenesis will promote the expansion of stem cells by preventing their differentiation. The compounds that we identify will be tested on cultured mouse and human ES cells for their effect on stem cell growth and differentiation. These experiments will identify compounds that promote stem cell self-renewal and will likely provoke future investigations to assess the potential therapeutic use of some of these compounds in promoting the replenishment of diseased tissues.
Role of miRNAs in human disease
We have identified particular miRNAs that are likely candidate genes for certain human diseases and are characterizing the expression profiles of these miRNAs, and in collaboration with appropriate laboratories, are performing DNA sequence analysis of patients in order to identify mutations in the miRNA gene that may contribute to the disease phenotype. Subsequently, we will identify the target mRNAs that are deregulated by abrogated miRNA function, and will generate mouse models of the disease by genetically targeting the disease-associated miRNAs.
Identification of miRNA target genes
Although much progress has been made into the identification of new miRNAs, much less is known about the function of individual miRNAs, and the identity of the genes whose expression they regulate. In order to address this we are developing a novel biochemical approach for the large-scale identification of miRNA target genes. We focus on those miRNAs that have been linked with disease, for example miRNAs with tumor suppressor or oncogene action.
PiRNAs: a new class of small non-coding RNA molecules
Members of the Argonaute family of proteins play a central role in "RNA silencing" pathways that regulate transcription, heterochromatin, genome integrity, and mRNA stability. Argonaute proteins are divided into two subfamilies, Ago and Piwi. Ago members are ubiquitously expressed and are required for posttranscriptional gene silencing. MicroRNAs and short-interfering RNAs of 22 nucleotides (nt) in length interact directly with Ago proteins and guide these complexes to target mRNAs of complementary sequence. The expression of the members of the Piwi sub-family is restricted to stem cells and germ cells. Recently, a novel class of abundant small RNAs known as Piwi-interacting RNAs (piRNAs) was identified in mammalian cells, that range in size from 26-31 nt. This exciting discovery raises several fundamental questions of significant biological and biomedical relevance; what is the function of piRNAs? How are piRNAs generated? What is the identity of Piwi-interacting proteins? Although piRNA biogenesis and function is not yet well understood, Piwi proteins are highly expressed during mouse spermatogenesis and are important for gamete formation. We hypothesize that Piwi proteins and associated small RNAs have additional stem cell functions in mammals. We therefore aim to biochemically define the composition of Piwi-containing ribonucleoprotein complexes in human and mouse stem cells, towards our goal of understanding the mechanism of piRNA biogenesis, the function of the Piwi-complexes, and their requirement for stem cell biology.
Gregory R. I.*, Yan K.*, Amuthan G., Chendrimada T., Doratotaj B., Cooch N. and Shiekhattar R. The Microprocessor complex mediates the genesis of microRNAs. Nature 2004;432:235-240. (* equal contribution).
Chendrimada T.*, Gregory R. I.*, Kumaraswamy E.*, Norman J., Cooch N., Nishikura K., and Shiekhattar R. TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature 2005;436:740-4. (* equal contribution).
Gregory R. I., and Shiekhattar, R. MicroRNA biogenesis and cancer. Cancer Research 2005;65:3509-12.
Gregory R. I., Chendrimada T., Cooch N., and Shiekhattar R. Human RISC couples microRNA maturation and posttranscriptional gene silencing. Cell 2005;123:631-40.
Gregory R. I., and Shiekhattar, R. MicroRNA biogenesis: Isolation and characterization of the Microprocessor complex. "MicroRNA Protocols", Methods in Molecular Biology 2006; 342, 33-47.
Chendrimada T., Finn K., Ji X, Baillat D, Gregory R. I., Liebhaber S, Pasquinelli A, and Shiekhattar R. MicroRNA silencing through RISC recruitment of eIF6. Nature 2007; 447:823-8.