Regulation of Pre-mRNA Splicing in Mammalian Cells
The majority of human genes are interrupted by non-coding sequences known as introns, whose removal is essential in the pathway of normal gene expression. It is estimated that up to 50% of point mutations responsible for human genetic diseases cause aberrant splicing which is linked to various forms of cancer. For instance, a mutation of the invariant adenosine residue within the branchpoint (in the intron 5) of the NF2 gene results in reduced usage of downstream exon and leads to neurofibromatosis type 2 (NF2), which is associated with tumors of the central nervous system such as schwannomas and ependymomas. Understanding the basic mechanisms of pre-mRNA branchpoint recognition is therefore highly relevant to the understanding of human diseases.
Removal of introns takes place within the spliceosome and involves two transesterification reactions. An adenosine embedded within the branchpoint sequence (BPS) of pre-mRNA plays a crucial role during both steps of splicing. One central and unresolved issue in pre-mRNA splicing is the molecular basis of branchpoint adensoine recognition and its activation as a nucleophile. We believe that key to understanding the spliceosome catalysis is determining the underlying networks of interactions that recognize adenosine as a branchpoint nucleotide. To achieve this objective, we are using a chemical genetics approach. Specifically, we generate model pre-mRNA substrates bearing modification of the chemical groups (in the RNA backbone, exocyclic base or the sugar) at a predetermined site, and then analyze the consequences of these modifications on the substrate recognition and catalytic properties of the spliceosome. Our ultimate goal is to develop a detailed understanding of the active site of the spliceosome.
Small Molecule Modulators of Cyclin-Dependent Kinases for Cancer Therapy
Cell growth and division is a highly regulated phenomenon, which is coordinated in the cell cycle. Cyclin-dependent kinases (CDKs) play a central role in cell cycle regulation, and aberrant activity of CDK has been associated with a variety of human cancers, including breast and prostate cancer. Thus, targeting CDK kinase activity has turned out to be the most productive strategy for the discovery and designing of novel anticancer molecules. With an aim to identify agents that may selectively inhibit CDK activity, we decided to screen a small molecule library comprising more than a hundred synthetic flavonoids. Flavonoids are naturally occurring polyphenols, which are known to exhibit antioxidant, antibacterial, antiviral, and anticancer activity. We observed that eight of these compounds were able to inhibit the proliferation of human MCF7 cells with an IC50 value between 1-10 µM. Significantly, at least two of these compounds mediated G1/S cell cycle arrest in MCF7 cells at concentrations of 3-10 µM (Fig. 1). Our long-term goal is to understand the molecular mechanisms by which these molecules assert their tumoricidal activities, and design analogues with more potent and selective anticancer activity.
Click to enlarge
