Interrogating PTB--Associated Splicing Factor's RNA Recognition Motif/RNA Binding-Mode / Krystal Haislop.

Format:

Book

Thesis/Dissertation

Author/Creator:

Haislop, Krystal, author.

Contributor:

University of Pennsylvania. Biochemistry and Molecular Biophysics, degree granting institution.

Language:

English

Subjects (All):

Biophysics.

Biochemistry.

Biochemistry and Molecular Biophysics--Penn dissertations.

Penn dissertations--Biochemistry and Molecular Biophysics.

Local Subjects:

Biophysics.

Biochemistry.

Biochemistry and Molecular Biophysics--Penn dissertations.

Penn dissertations--Biochemistry and Molecular Biophysics.

Physical Description:

1 online resource (111 pages)

Distribution:

Ann Arbor : ProQuest Dissertations & Theses, 2022

Contained In:

Dissertations Abstracts International 84-02B.

Place of Publication:

[Philadelphia, Pennsylvania] : University of Pennsylvania, 2022.

Language Note:

English

Summary:

PTB-associated splicing factor (PSF) is a multifunctional nucleic acid binding protein vital for cell survival. PSF plays several roles throughout the cell, yet many aspects of PSF's mechanisms of interacting remain elusive. While protein and nucleic acid binding partners of PSF have been identified, binding consensus sequences reported in the literature vary. Previous studies in the Lynch Laboratory have found that PSF's second RNA recognition motif (RRM2) is necessary and sufficient for binding, but this binding can be occluded by interacting with cofactor protein TRAP150. Additionally, binding is dependent on phosphorylation of PSF in T-cells. Identifying the interaction interfaces of PSF/RNA and PSF/TRAP150 yields insight into the regulatory mechanism governing RNA-binding. First, I utilize biophysical techniques to overcome challenges characterizing PSF/RNA. The smallest RNA to bind with low/modest affinity is 65-nucleotides long, making the protein/RNA pair non-ideal for crystallization or NMR experiments. PSF has long N- and C-terminal unstructured regions that aid in PSF's functional aggregation, creating another challenge for biophysical characterization. Here I use several biophysical techniques to characterize PSF's dynamics and binding interfaces. Mass spectrometry experiments indicate PSF contacts RNA using 30-60% of the accessible surface area. Interestingly, a charged hydrophobic loop region when mutated from "DDRGR" to "AAAAA" increases affinity for ESS-RNA 10-fold. A neighboring residue Lysine-413 also increases affinity for RNA when mutated to alanine. Arginine-474 and Threoinine-485 within the NOPS domain lose their ability to bind RNA when mutated. It is not clear whether these residues affect local conformation of playing a direct role in binding. Additionally, I identify hundreds of PSF-dependent alternative polyadenylation changes and begin characterization of PSF/TRAP150 and phosphorylated PSF by HDX-MS. Together, these results indicate PSF interacts with RNA using a large portion of the surface accessible area to do so, and PSF uses a loop in RRM2 to aid in RNA-interaction.

Notes:

Source: Dissertations Abstracts International, Volume: 84-02, Section: B.

Advisors: Lynch, Kristen W.; Committee members: Gupta, Kushol; Rhoades, Elizabeth; Speicher, David W.; Weitzman, Matthew.

Department: Biochemistry and Molecular Biophysics.

Ph.D. University of Pennsylvania 2022.

Local Notes:

School code: 0175

ISBN:

9798837502286

Access Restriction:

Restricted for use by site license.