Overview

The ability to silence genes in mammalian cells through 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. MicroRNAs are encoded by endogenous genes and regulate over half of all genes in mammalian cells. They regulate gene expression at the stage of translation and mRNA stability. We are studying this translational repression, particularly in relationship to cellular stress. We are analyzing the nature of gene regulation by small RNAs such as microRNAs in embryonic stem cells and in lymphocytes. We are also using RNAi technology to identify specific proteins important for regulation as a consequence of Ras activation of alternative RNA splicing of the CD44 gene.

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 is largely 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 have investigated the use of siRNAs to silence genes in a variety of cell lines. To stably produce gene silencing in mammalian cells in culture, 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 are thought to regulate gene expression primarily by inhibiting accumulation of protein. Some 250-350 conserved miRNA genes are encoded in the human genome. An indication that siRNAs function through the miRNA-pathway is the observation that these RNAs will inhibit the translation of a reporter gene, which contains multiple partially complementary target sites (see Figure 2). 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. Some studies have related miRNA silencing to the activities of P granules, sites of mRNA degradation in cells. We have recently shown that miRNAs are associated with stress granules in mammalian cells. The latter cytoplasmic components also contain Argonaute proteins, factors important for silencing by miRNA.  We are investigating the role of stress granules in gene regulation by miRNAs. miRNAs 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.

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. About half of all mammalian genes have tandem polyadenylation sites. The shorter 3’ UTRs in proliferative cells are probably generated by cleavage and polyadenylation at the most upstream polyA sites. Alternatively, genes expressed in quiescent cells, the most frequent cell state in most tissue, have long 3’ UTRs. This predicts that gene regulation through recognition of 3’ UTR sequences by microRNAs and RNA binding proteins is more extensive in quiescent cells.

RNA Splicing: Gene sequences important for accurate splicing of the nuclear precursor to mRNAs are 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.

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 normal cells. Ten internal exons are variably included in the tumor-associated isoforms and Ras activation stimulates their expression. In a positive feedback loop, these CD44 isoforms also activate the Ras signaling pathway. One function of this positive feedback loop is to sustain Ras activation to allow cells to cross the transition from GI to S phase.

Several RNA binding proteins have been shown to be important for inclusion of the variable exons of CD44. 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. Signaling pathways controlling alternative RNA splicing are being investigated using siRNA specific gene silencing methods.

Selected Publications

Dennis, L.M, Gill, M.E., Zheng, G.X.Y., Medeiros, L.A., Markoulaki, S., Seila, A.C., Welstead, G.G., Fu, D.D., Houbaviy, H.B., Page, D.C., Sharp, P.A., and Jaenisch, R. The miRNA cluster, miR-290 through miR-295, is an important regulator of early embryogenesis and female germ cell development. Submitted (2008).

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).

White, 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. Submitted (2008).

Seila, A.C. and Sharp, P.A. Small RNAs tell big stories in Whistler. Meeting Report on Keystone Symposium on RNAi, microRNA and non-coding RNA, March 25-30, 2008. Nature Cell Biology 10, 630-633 (2008).

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).

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).

Ebert, M.S., Neilson, J.R., and Sharp, P.A. MicroRNA sponges: Competitive inhibitors of small
RNAs in mammalian cells. Nature Methods 4, 721-726 (2007).

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