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

Abstract

The 7SK ribonucleoprotein (RNP) is a critical regulator of eukaryotic transcription. Recently, RNA binding motif 7 (RBM7) containing 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 found that RRM constructs containing extended C-termini have increased solubility compared to a minimal RRM construct, although these constructs aggregate in a temperature and concentration-dependent manner. Using solution NMR dynamics experiments, we identified additional structural features observed previously in crystal but not in solution structures. To identify potential RBM7-7SK RNA binding sites, we analyzed deposited data from in cellulo crosslinking experiments and found that RBM7 primarily crosslinks 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.