Presentation Title

Characterizing the Role of Linker 2 Region in the Splicing Activity of an RNA Binding Protein

Faculty Mentor

Niroshika Keppetipola

Start Date

23-11-2019 8:45 AM

End Date

23-11-2019 9:30 AM

Location

56

Session

poster 2

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Polypyrimidine tract binding proteins PTBP1 and PTBP2 are paralogous RNA binding proteins. They have similar tertiary structures with four RNA binding domains, connected by three linker regions and an N-terminal region. Their primary structures share 74% identity, with 80% or greater identity over the RBD regions, 100% identity in the Linker 3 region but less identity over other regions. PTBP1 and PTBP2 have non-overlapping tissue-specific expression patterns; PTBP1 is expressed near ubiquitously and PTBP2 is expressed in differentiating neurons and myocytes. The two proteins regulate over-lapping and non-overlapping sets of target exons. Their distinct expression patterns coupled to the non-overlapping splicing targets play a critical role during neuronal development and maturation. PTBP1 acts as a suppressor of many neuronal regulated exons but PTBP2 does not. It is yet to be understood how these paralogous proteins exert different splicing outcomes. We discovered that PTBP1 and PTBP2 are post-translationally phosphorylated. The Thre298 and Ser308 residues in the Linker 2 region of PTBP2 are significantly phosphorylated while PTBP1 does not have residues at these positions that can undergo phosphorylation. A PTBP1-PTBP2 chimera that contained the PTBP1 Linker 2 region in a PTBP2 protein demonstrated PTBP1-like splicing activity. It is possible that reversible phosphorylation at Thr298 and Ser308 plays a role in PTBP2’s neuronal specific splicing activity. To test this, we generated S308A and T298A mutants using recombinant DNA technology and assayed the mutant for protein expression and splicing activity in vivo in mouse neuro 2A cells. Our results indicate the mutants are well expressed. We are currently assaying the mutants for splicing activity. Data from these experiments will reveal the role of Thr298 and Ser308 in PTBP2 splicing activity.

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Nov 23rd, 8:45 AM Nov 23rd, 9:30 AM

Characterizing the Role of Linker 2 Region in the Splicing Activity of an RNA Binding Protein

56

Polypyrimidine tract binding proteins PTBP1 and PTBP2 are paralogous RNA binding proteins. They have similar tertiary structures with four RNA binding domains, connected by three linker regions and an N-terminal region. Their primary structures share 74% identity, with 80% or greater identity over the RBD regions, 100% identity in the Linker 3 region but less identity over other regions. PTBP1 and PTBP2 have non-overlapping tissue-specific expression patterns; PTBP1 is expressed near ubiquitously and PTBP2 is expressed in differentiating neurons and myocytes. The two proteins regulate over-lapping and non-overlapping sets of target exons. Their distinct expression patterns coupled to the non-overlapping splicing targets play a critical role during neuronal development and maturation. PTBP1 acts as a suppressor of many neuronal regulated exons but PTBP2 does not. It is yet to be understood how these paralogous proteins exert different splicing outcomes. We discovered that PTBP1 and PTBP2 are post-translationally phosphorylated. The Thre298 and Ser308 residues in the Linker 2 region of PTBP2 are significantly phosphorylated while PTBP1 does not have residues at these positions that can undergo phosphorylation. A PTBP1-PTBP2 chimera that contained the PTBP1 Linker 2 region in a PTBP2 protein demonstrated PTBP1-like splicing activity. It is possible that reversible phosphorylation at Thr298 and Ser308 plays a role in PTBP2’s neuronal specific splicing activity. To test this, we generated S308A and T298A mutants using recombinant DNA technology and assayed the mutant for protein expression and splicing activity in vivo in mouse neuro 2A cells. Our results indicate the mutants are well expressed. We are currently assaying the mutants for splicing activity. Data from these experiments will reveal the role of Thr298 and Ser308 in PTBP2 splicing activity.