Overview

MicroRNAs (miRNAs) are encoded by endogenous genes and regulate over half of all genes in mammalian cells. They regulate gene expression at the stages of translation and mRNA stability. Developing methods to physically identify the target mRNAs for particular miRNAs is on going. The surprising recent finding that expression of certain microRNAs can induce a pluripotent stem cell indicates that this mechanism of cytoplasmic regulation rivals that of transcription factors in the nucleus. RNA interference (RNAi) has dramatically expanded the possibilities for genotype/phenotype analyses in cell biology. Investigations into the mechanisms responsible for the activities of short interfering RNAs (siRNAs) are underway with the objective of increasing their effectiveness in gene silencing. Delivery of siRNAs by nanoparticles to silence genes in tumors is being tested in ovarian tumor models. High throughput sequencing of RNA populations revealed the generation of small RNAs in divergent transcription in mammalian cells. The role of this pervasive transcription from the anti-sense strand is under investigation. It is likely that these anti-sense transcripts are unstable because, in contrast to the sense transcript, they are not recognized by the certain RNA splicing factors. The same high throughput technology allows definition of alternatively spliced isoforms. Shifts in isoforms are common in cancer versus normal cells. Also, recent results from other labs have suggested that chromatin structure is related to control of alternative splicing. We are investigating these processes and, in particular, the relationship between elongation of transcription, RNA splicing and chromatin modifications.

Research Summary

RNA Interference: RNAi was first identified as a post-transcriptional response to exogenous double-stranded RNA (dsRNA) introduced into the nematode worm, C. elegans, and many aspects of RNAi are conserved from fungi to plants to mammals. The pathway is triggered when long dsRNA encounters the RNaseIII enzyme, Dicer, a cytoplasmic enzyme that cleaves the dsRNA to produce short interfering RNAs (siRNAs). One strand of the siRNA is incorporated into the effector complex of RNAi, the RNA-induced Silencing Complex (RISC). The short RNA guides RISC to target mRNA and catalyzes an endonucleolytic cleavage, resulting in a post-transcriptional silencing of gene expression (Figure 1). We are investigating the use of siRNAs to silence genes in a variety of cell types and to treat diseases such as cancer. We hope by better understanding the activities of siRNAs in mammalian cells these gene silencing processes can be made more effective.

MicroRNAs (21-22 nt) are processed from hairpin RNAs encoded by cellular DNA and regulate gene expression primarily by inhibiting translation and promoting mRNA degradation. Some 250-350 conserved miRNA genes are encoded in the human genome (see Figure 2). siRNAs function through the miRNA-pathway and these RNAs will inhibit the translation of a reporter gene that contains multiple partially complementary target sites. We have developed methods for identifying the targets of miRNAs and we surprisingly found that mRNAs appear to be bound to components of the miRNP in the absence of miRNAs. miRNA regulation is not essential for survival, not even for some tumorigenic properties of mammalian cells. We have recently isolated a sarcoma tumor cell line that is null for dicer, devoid of miRNAs, and yet can produce a tumor in vivo. However this cell line is very sensitive to stresses.

Small RNAs are known to regulate developmental transitions in many biological systems. The differentiation of embryonic stem (ES) cells is easily induced and has been well studied. We have cloned miRNAs from undifferentiated and differentiated cultures of ES cells. Surprisingly, we found a cluster of six miRNA genes specifically expressed in undifferentiated ES cells. A homologous cluster has been identified in human embryonic stem cells. This cluster is only expressed in embryonic tissue in mouse and we have recently found, in collaboration with the Jaenisch laboratory, that females with deletions of this cluster are defective in the generation of germ cells. Embryonic stem cells null for this cluster are more sensitive to induction of cell death and over express genes that activate this process.

We have recently reported that divergent transcription is common of the promoter sites for genes in embryonic stem cells. These promoters have an RNA polymerase initiated in the sense direction immediately downstream of the transcription start site and a second polymerase initiated in the antisense direction, about 250 base pairs upstream. The evidence for this structure is multifold. It includes the identification of small RNAs from these two regions of many promoters, detection of small RNAs by Northerns and mapping of RNA polymerase and modifications of chromatin in these regions. This research has been done in collaboration with Professor Rick Young. Surprisingly, the anti-sense polymerase is controlled by elongation processes very similar to those of sense polymerase. For example, both require P-TEFb for elongation beyond about 30 nts. The nature of factors or sequences that differentiate the effective elongation of the polymerase in the sense direction as compared to the ineffective elongation in the anti-sense direction remains to be identified.

RNA Splicing: Gene sequences important for accurate splicing of the nuclear precursor to mRNAs are commonly conserved during evolution. We are using computational methods to identify, by comparison of genomic sequences from multiple organisms, intron and exon sequences which are important for accurate splicing and for control of alternative RNA splicing. The cell surface protein CD44 is expressed as a variety of isoforms in tumor and activated cells but is present in a constitutive form in quiescent cells. These isoforms influence the cells’ motility, invasiveness and recognition of extracellular factors. Accordingly, shifts in the prevalence of these isoforms occur as tumor cells become more invasive such as in the epithelial to mesenchymal transition. RNA binding proteins and signaling pathways controlling alternative RNA splicing of CD44 are being investigated using high throughput sequencing methods to define transcriptomes. We are also investigating the relationship between chromatin structure and alternative RNA splicing.

Selected Publications

Edbauer, D., Neilson, J., Foster, K.A., Wang, C.-F., Seeburg, D.P., Batterton, M.N., Tada, T., Dolan, B.M., Sharp, P.A., and Sheng, M. Regulation of synaptic structure and function by FMRP-associated microRNAs miR-125b and miR-132. Neuron 65, 373-384 (2010).

Rahl, P.B. Lin, C.Y., Seila, A.C., Flynn, R.A., McCuine, S., Burge, C.B., Sharp, P.A., and Young, A. c-Myc regulates transcriptional pause release. Cell 141, 432-445 (2010). PMCID: PMC2864022

Leung, A.K.-L. and Sharp, P.A. MicroRNA functions in stress responses. Mol Cell. 40, 205-215 (2010). PMCID: PMC2996264

Ebert, M.S. and Sharp, P.A. MicroRNA sponges: progress and possibilities. RNA 16, 2043-2050 (2010). PMCID: PMC2957044

Ebert, M.S. and Sharp, P.A. Emerging roles for natural microRNA sponges. Curr Biol. 20 R858-R861 (2010).

Creyghton, M., Cheng, A., Welstead, G.G., Kooistra, T., Carey, B., Sharp, P.A., Steine, E., Hanna, J., Lodato, M., Frampton, G., Boyer, L., Young, R., and Jaenisch, R. Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc. Natl Acad of Sci USA 107, 21931-21936 (2010). PMCID: PMC3003124.

Singh, N., Agrawal, A., Leung, A.K., Sharp, P.A., and Bhatia, S.N. Effect of nanoparticle conjugation on gene silencing by RNA interference. J. Am. Chem. Soc. 132, 8241-8243 (2010). PMCID: PMC2968757

Goldberg, M.S., Xin, D., Ren, Y., Orsulic, S., Bhatia, S.N., and Sharp, P.A. Nanoparticle-mediated delivery of siRNA targeting Parp1 extends survival of mice bearing tumors derived from Brca1-deficient ovarian cancer cells. Proc Natl Acad Sci USA 108, 745-750 (2010). PMCID: PMC3021044

Leung, A.K.-L., Young, A.G., Bhutkar, A., Zheng, G.X., Bosson, A.D., Nielsen, C.B., and Sharp P.A. Genome-wide identification of Ago2 binding sites from mouse embryonic stem cells with and without mature microRNAs. Nat Struct Mol Biol. 18, 237-244 (2011). PMCID: PMC3078052

Flynn, R.A., Almada, A.E., Zamudio, J.R., and Sharp, P.A. Antisense RNA polymerase II divergent transcripts are P-TEFb-dependent substrates for the RNA exosome. Proc. Natl. Acad. Sci USA 108, 10460-10465 (2011). PMCID: PMC3127934

Zheng, G., Ravi, A., Calabrese, J.M., Medeiros, L.A., Kirak, O., Dennis, L.M., Jaenisch, R., Burge, C.B., and Sharp, P.A. A latent pro-survival function for the Mir-290-295 cluster in mouse embryonic stem cells. PLoS Genetics 7, e1002054 (2011). PMCID: PMC3088722

Meenhuis, A., van Veelen, P.A., van den Berge, I.J., Sun, S.M., Taskesen, E., Stern, P., de Ru, A.H., van Adrichem, A.J., Demmers, J., Jongen-Lavrencic, M., Löwenberg, B., Touw, I.P., Sharp, P.A., and Erkeland, S.J. MiR-17/20/93/106 promote hematopoietic cell expansion by targeting sequestosome 1-regulated pathways in mice. Blood 118, 916-925 (2011). PMCID Journal in process.

Zheng, G., Ravi, A., Gould, GM, Burge, CB, and Sharp, PA. Genome-wide signature of a novel rapidly expanded microRNA cluster in mouse. Proc Natl Acad Sci USA, in press. PMCID Journal in process.

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