Presentation Title

Understanding the role of post-translational modifications on the splicing activity of two related RNA binding proteins

Start Date

November 2016

End Date

November 2016

Location

HUB 302-#7

Type of Presentation

Poster

Abstract

Several human diseases including cancer and Alzheimer’s disease are associated with alternative splicing, one mechanism by which a diverse amount of proteins are produced from a single gene in eukaryotic cells. Alternative splicing is regulated in part by RNA Binding Proteins. The Polypyrimidine Tract Binding Protein (PTBP) is an RNA binding protein that has many functions including alternative splicing regulation and mRNA localization. The PTBP gene family has 3 paralogs; PTBP1, PTBP2 and PTBP3. The paralogs have high primary structure identity and similar domain organization yet has tissue specific expression patterns and different splicing effects on certain target exons. In our study, we focus on paralogs PTBP1 and PTBP2. PTBP1 is expressed near ubiquitously, however is absent in neurons and muscle cells. PTBP2 is expressed in neurons. The two proteins have 74% primary structure identity however exert different splicing outcomes on certain regulated exons. The levels of the two proteins change during neuronal differentiation and maturation and this change in protein concentration is critical for neuronal development. Recent studies indicate that multiple determinants over the regions of the two proteins dictate their differential splicing activity. Genome-wide mass spectrometry studies indicate that the two proteins are post-translationally modified. The hypothesis underlying our studies is that the modifications dictate the differential splicing activity of the two proteins. To test this, we have overexpressed PTBP1 and PTBP2 in HEK293T and mouse N2A cells and assayed via Western Blot. We have probed for protein phosphorylation via Phos-Tag Gel Electrophoresis. We have probed for ubiquitiantion and acetylation using modification specific antibodies via Western Blot. We have purified Flag-tagged PTBP1 and PTBP2 via Flag-Immunoprecipitation for mass spectrometry analysis for post-translational modifications. Preliminary results indicate that the proteins are modified in HEK293T, N2A cells and that they have different interacting partner proteins.

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Understanding the role of post-translational modifications on the splicing activity of two related RNA binding proteins

HUB 302-#7

Several human diseases including cancer and Alzheimer’s disease are associated with alternative splicing, one mechanism by which a diverse amount of proteins are produced from a single gene in eukaryotic cells. Alternative splicing is regulated in part by RNA Binding Proteins. The Polypyrimidine Tract Binding Protein (PTBP) is an RNA binding protein that has many functions including alternative splicing regulation and mRNA localization. The PTBP gene family has 3 paralogs; PTBP1, PTBP2 and PTBP3. The paralogs have high primary structure identity and similar domain organization yet has tissue specific expression patterns and different splicing effects on certain target exons. In our study, we focus on paralogs PTBP1 and PTBP2. PTBP1 is expressed near ubiquitously, however is absent in neurons and muscle cells. PTBP2 is expressed in neurons. The two proteins have 74% primary structure identity however exert different splicing outcomes on certain regulated exons. The levels of the two proteins change during neuronal differentiation and maturation and this change in protein concentration is critical for neuronal development. Recent studies indicate that multiple determinants over the regions of the two proteins dictate their differential splicing activity. Genome-wide mass spectrometry studies indicate that the two proteins are post-translationally modified. The hypothesis underlying our studies is that the modifications dictate the differential splicing activity of the two proteins. To test this, we have overexpressed PTBP1 and PTBP2 in HEK293T and mouse N2A cells and assayed via Western Blot. We have probed for protein phosphorylation via Phos-Tag Gel Electrophoresis. We have probed for ubiquitiantion and acetylation using modification specific antibodies via Western Blot. We have purified Flag-tagged PTBP1 and PTBP2 via Flag-Immunoprecipitation for mass spectrometry analysis for post-translational modifications. Preliminary results indicate that the proteins are modified in HEK293T, N2A cells and that they have different interacting partner proteins.