UC Santa Cruz
A Pipeline for Tagging snRNP Associated Proteins in HeLa
- Author(s): Kim, John
- Advisor(s): Jurica, Melissa
- et al.
The spliceosome is a dynamic eukaryotic macromolecular complex responsible for catalysis of pre-mRNA splicing. High quality processing of a pre-mRNA by removal of non-coding introns and ligation of exons is essential to preserve the accuracy of a mature RNA transcript that will undergo eventual translation into a functional protein. Given the irresolute nature of the spliceosome, much of the machinery underlying the structural rearrangements that occur throughout the splice cycle have yet to be fully characterized. Thus far, biochemical characteristics of spliceosome assembly in humans have been studied by complexes purified by in-vitro reconstitution with HeLa nuclear extract and a transcribed radiolabeled pre-mRNA substrate. While in-vitro reconstitution has been a standard for purification of both human and yeast spliceosomes, studies in yeast have also been able to utilize endogenous protein tags to study the physical nature of spliceosome intra-interactions. Here we have re-purposed a RMCE protocol from Khandelia et al. to endogenously express V5-tagged snRPB2, a core component of the U2 snRNP, in HeLa cells. Our tagged construct was integrated into the HeLa cell genome by Cre-Lox recombination and construct expression is regulated by a TET-ON system. Selected HeLa cells were then induced to express V5-tagged snRPB2 for eventual purification of the tagged protein and its endogenous interactions by co-immunoprecipitation (Co-IP). Mass spectrometry data revealed the Co-IP eluent contains all the components of an endogenously assembled U2snRNP from active HeLa nuclear extract. Altogether, these preliminary results open the door for a pipeline to study biochemical characteristics of mammalian spliceosomal complexes purified via endogenously tagged proteins.