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), 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 abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/491737v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@10ac49org.highwire.dtl.DTLVardef@9f1de4org.highwire.dtl.DTLVardef@63985eorg.highwire.dtl.DTLVardef@a870a7_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIExtending the RBM7 RRM C-terminus improves protein expression and solubility C_LIO_LIThe RBM7 RRM interacts weakly with 7SK stem-loop 3 single-stranded RNA C_LIO_LISolution state NMR dynamics measurements support presence of additional {beta}4 strand C_LI