Overview

 RNA interference (RNAi) has dramatically expanded the possibilities for genotype/phenotype analysis in cell biology.  Investigations into the mechanisms responsible for the activities of short interfering RNAs (siRNAs) are ongoing 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. 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.  Physically identifying the target mRNAs for particular miRNAs is of great interest. We are also investigating the relationship between gene regulation by miRNAs and cellular stress. The roles of small RNAs in embryonic stem cells and in lymphocytes are being investigated.  We are also studying the nature of specific proteins important for regulation of alternative RNA splicing of the CD44 gene in normal and tumor cells.

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. To stably produce gene silencing, DNA sequences encoding a 21 bp inverted repeat or hairpin corresponding to an active siRNA can be inserted downstream of a promoter in a retroviral vector and used to infect cells. 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 are exploiting this finding to study the mechanism of translational inhibition by miRNAs and to develop a purification protocol for identifying the targets of miRNAs. miRNA regulation is not essential for survival nor 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.

Many systems with deletions of particular miRNA genes are more sensitive to stresses than their normal counterparts. We have recently shown that miRNAs are associated with stress granules that form under these conditions in mammalian cells. The latter cytoplasmic components also contain Argonaute proteins, factors important for silencing by miRNA. We have recently discovered in collaboration with Professor Paul Chang’s laboratory that formation of stress granules are dependent upon modifications of cytoplasmic RNA binding proteins by Poly(ADP-ribose). In fact, inhibiting modification of these proteins by Poly(ADP-ribose) modulates the level of gene regulation by miRNAs. 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, all within a segment of 2.2 kb, 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 are further investigating two fascinating findings. In collaboration with Chris Burge, we have characterized mRNA populations from quiescent and proliferating T cells. Surprisingly, many mRNAs expressed in proliferating T cells have shorter 3’ UTRs than those in quiescent cells. When publically available databases were examined, the same appeared to be true for most normal and tumor cells. This shift is probably significant since about half of all mammalian genes have tandem polyadenylation sites. The shorter 3’ UTRs in proliferative cells are probably generated by enhanced cleavage and polyadenylation rates at the upstream polyA sites. Since most miRNAs and some well-studied RNA binding proteins target sequences in 3’UTRs, this predicts that gene regulation by these agents is more extensive in quiescent cells.
The second finding is 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 is part of a collaboration with Professor Rick Young. The processes generating divergent transcription at promoters and its significance in gene regulation is of interest.

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. Ten internal exons are variably included in the tumor-associated isoforms. 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. Several RNA binding proteins have been shown to be important for inclusion of the variable exons of CD44. In addition, the SRm160 protein, which does not directly bind RNA but is a splicing co-factor, is also important for the alternative splicing of CD44 isoforms. RNA binding proteins and signaling pathways controlling alternative RNA splicing of CD44 are being investigated using siRNA specific gene silencing methods.

Selected Publications

Sandberg, R., Neilson, J.R., Sarma, A., Sharp, P.A., and Burge, C.  Widespread evasion of posttranscriptional regulation associated with proliferation. Science 320, 1643-1647 (2008). PMCID: PMC2587246

Seila, A.C., Calabrese, J.M., Levine, S.S., Yeo, G.W., Rahl, B., Young, R.A., and Sharp P.A.  Divergent transcription from active promoters. Science 322, 1849-1851 (2008). NIHMSID: 94606

Marson, A., Levine, S.S., Cole, M.F., Frampton, G.M., Brambrink, T., Johnstone, S., Guenther, M.G., Johnston, W.K., Wernig, M., Newman, J., Calabrese, M., Dennis, L.M., Volkert, T.L., Gupta, S., Love, J., Hannett, N., Sharp, P.A., Bartel, D.P., Jaenisch, R., and Young, R.A.  Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells.  Cell 134, 521-533 (2008). PMCID: PMC2586071

Ventura, A., Young, A.G., Winslow, M.M., Lintault, L., Meissner, A., Erkeland, S.J., Newman, J., Bronson, R.T., Crowley, D., Stone, J.R., Jaenisch, R., Sharp, P.A. and Jacks, T.  Targeted deletion reveals essential and overlapping functions of the miR-17~92 family of miRNA clusters. Cell 132, 875-886 (2008).  PMCID: PMC2323338

Kumar, M.S., Erkeland, S.J., Pester, R.E., Chen, C. Y., Ebert, M.S, Sharp, P.A., Jacks, T.   Suppression of non-small cell lung tumor development by the let-7 microRNA family.  Proc. Natl. Acad. Sci., USA 105, 3903-3908 (2008).  PMCID: PMC2268826

Sharp, P.A.  The Centrality of RNA (Leading Edge Essay).  Cell 136, 577-580 (2009).

Agrawal, A., Min, D.H., Singh, N., Zhu, H., Birjiniuk, A., von Maltzahn, G., Harris, T.J., Xing, D., Woolfenden, S., Sharp, P.A., Charest, A., and Bhatia, S.N. (2009). Functional delivery of siRNA in mice using dendriworms.  ACS Nano, in press (2009).

Ravi, A.R., Kumar, M.S., Chin, C., Jacks, T., and Sharp, P.A. Viability of a somatic cell type lacking microRNAs.  Submitted (2009).

Leung, A.K.L., Vyas, S., Sharp, P.A., and Chang, P.  Poly(ADP-ribose) regulates microRNA activity and is required for stress granule integrity.  Submitted (2009).

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