Sharp Lab
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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

Non-coding RNAs::

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 1). 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 the RNP complex containing 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.

We have recently reported that divergent transcription is common of promoter sites for genes in embryonic stem cells (see Figure 2). 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 Richard 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 50 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.

Long non-coding RNAs (lncRNAs) have been described from analysis of deep RNA sequencing from many types of mammalian cells. Comparable RNA species have also been reported from sequencing data of yeast and Drosophila. Recent analysis of several large data sets of RNA sequences expressed in embryonic stem cells shows that a majority of long non-coding RNAs originated from initiation sites that are divergent from known protein-encoding genes or sites with chromatin marks indicating enhancer elements. Thus, synthesis of some long non-coding RNAs is probably a manifestation of general transcriptional processes. However, these lncRNAs could function in regulation of genes in cis to the site of transcription or in trans at other sites in the genome. In the latter case, the lncRNAs would probably need to be more abundant then the 1-2 copies per cells for most divergent transcripts.


(Click figures for higher resolution images) The many roles of RNA in the cell.

Figure 1
Figure 1

Figure 2
Figure 2

 

RNA Splicing:

Gene sequences important for accurate splicing of the nuclear precursors 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.

 

MIT Koch Institute for Integrative Cancer Research at MIT