Exploring dual DNA/RNA binding patterns in the FUBP family of nucleic acid binding proteins

Authors

• CH Danan
• M Hafner

Abstract

DNA- and RNA-binding proteins (DRBPs) are a largely unexplored subset of nucleic acid binding proteins that exhibit functionally significant binding to both DNA and RNA. Despite their low profile, recent evidence suggests that DRBPs comprise a critical class of proteins that could play roles in transcription, post-transcriptional gene regulation, translation, and telomere maintenance. The Far Upstream Element (FUSE) binding protein (FUBP1) was first identified binding single stranded DNA upstream of the oncogene Myc. FUBP1 belongs to a family of three paralogs (FUBP1-3) that share extensive sequence identity, including four conserved K-homology (KH) domains, nucleic acid binding domains with demonstrated affinity for ssDNA as well as ssRNA and protein. FUBP1 and FUBP3 have typically been described as ssDNA binding, while it is suggested that FUBP2 (gene symbol: KHSRP) is predominantly RNA-binding. We are using systems biology techniques to construct a single complete account of the FUBP family as both DNA and RNA regulators. Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) demonstrated that FLAG/HA-tagged KHSRP and FUBP1 exhibit comparable affinity for RNA in HEK293 cells. Analysis of KHSRP in vivo has also yielded functions inconsistent with the literature. Small interfering RNA knockdown followed by RNA sequencing in HEK293 cells showed that KHSRP stabilizes target transcripts. And immunoprecipitation of KHSRP followed by mass spectrometry suggests that KHSRP predominantly interacts with the DNA binding protein ZAP3. We plan to expand these and other approaches to all members of the FUBP family to uncover roles for FUBP proteins as regulators at the interface of DNA and RNA.

Scientific Focus Area: Molecular Biology and Biochemistry

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