C-terminal determinants for RNA binding motif 7 protein stability and RNA recognition

Abstract

AbstractThe 7SK ribonucleoprotein (RNP) is a critical regulator of eukaryotic transcription. Recently, RNA binding motif 7 (RBM7), which contains an RNA recognition motif (RRM), was reported to associate with 7SK RNA and core 7SK RNP protein components in response to DNA damage. However, little is known about the mode of RBM7-7SK RNA recognition. Here, we recombinantly expressed and purified RBM7 RRM constructs and found that constructs containing extended C-termini have increased solubility and stability compared to shorter constructs. To identify potential RBM7-7SK RNA binding sites, we analyzed deposited data from in cellulo crosslinking experiments and found that RBM7 crosslinks specifically to the distal region of 7SK stem-loop 3 (SL3). Electrophoretic mobility shift assays and NMR chemical shift perturbation experiments showed weak binding to 7SK SL3 constructs in vitro. Together, these results provide new insights into RBM7 RRM folding and recognition of 7SK RNA.Graphical abstractHighlightsExtending the RBM7 RRM C-terminus improves protein expression and solubilityThe RBM7 RRM interacts weakly with 7SK stem-loop 3 single-stranded RNASolution state NMR dynamics measurements support presence of additional β4’ strand