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Ronald J. Patterson, Ph.D.

Research

RNA splicing is a basic biochemical process in which non-coding nucleotide sequences (introns) are removed from pre-mRNA transcripts and coding sequences (exons) are ligated to form mature mRNA. This complex series of events is orchestrated by a macromolecular "machine" (the spliceosome) that contains several small nuclear RNA protein complexes (snRNPs) and numerous protein splicing co-factors. We have documented that galectin-1 (gal-1) and galectin-3 (gal-3) are novel splicing factors using a cell-free splicing assay. The key findings are:

• Both galectins are found in nuclei and nuclear extracts capable of in vitro splicing
• Depletion of both galectins from nuclear extracts abolishes splicing activity and blocks spliceosome assembly at the H/E complex
• Addition of either galectin (as a recombinant protein) restores both splicing activity and active spliceosome formation
• Both galectins co-localize with other known splicing factors in nuclear speckles.

Recently, we have identified a nuclear partner for these galectins using a yeast two hybrid genetic screen. This partner, Gemin4, is a member of the survival of motor neurons (SMN) complex implicated in supplying or recycling snRNPs to the spliceosome. In support of this suggestion, the addition of the amino terminal domain of gal-3 or the carboxyl terminal 50 amino acids of Gemin4 to a splicing reaction acts in a dominant negative fashion and inhibits splicing.

Using immunoprecipitation criteria, we have shown that gal-1 is a spliceosomal component. In these experiments, we have shown that antibodies to gal-1 immunoprecipitate:

• Splicing substrate pre-mRNA, splicing intermediates and products (interestingly, gal-1 appears to be preferentially found in complexes containing the excised intron lariat)
• Pre-spliceosomal complexes
• Known spliceosomal proteins and snRNPs
• The SMN polypeptide

Several major questions pertaining to the role of galectins in splicing remain unresolved:

• Is the carbohydrate-binding activity of the galectins separate from their splicing activity?
• Are there other partners for the galectins in the splicing pathway?
• Are galectins associated with pre-mRNAs in vivo?
• Do the galectins play a role in mRNA-related processes in the cytoplasm?

A variety of techniques in cell biology, nucleic acid biochemistry and molecular biology are used in these studies. The research is done in collaboration with Dr. John Wang in the Department of Biochemistry and Molecular Biology at Michigan State.