MRNIP is a replication fork protection factor.

The remodeling of stalled replication forks to form four-way DNA junctions is an important component of the replication stress response. Nascent DNA at the regressed arms of these reversed forks is protected by RAD51 and the tumor suppressors BRCA1/2, and when this function is compromised, stalled forks undergo pathological MRE11-dependent degradation, leading to chromosomal instability. However, the mechanisms regulating MRE11 functions at reversed forks are currently unclear. Here, we identify the MRE11-binding protein MRNIP as a novel fork protection factor that directly binds to MRE11 and specifically represses its exonuclease activity. The loss of MRNIP results in impaired replication fork progression, MRE11 exonuclease-dependent degradation of reversed forks, persistence of underreplicated genomic regions, chemosensitivity, and chromosome instability. Our findings identify MRNIP as a novel regulator of MRE11 at reversed forks and provide evidence that regulation of specific MRE11 nuclease activities ensures protection of nascent DNA and thereby genome integrity.

Detection of double-stranded RNA-protein interactions by methylene blue-mediated photo-crosslinking.

Double-stranded(ds) RNA-binding proteins have diverse functions in the cell. An obstacle to investigating the interactions between these proteins and dsRNA is the relative inefficiency of traditional UV-crosslinking methods for extended regions of dsRNA. We have therefore developed an alternative procedure for RNA-protein photo-crosslinking that efficiently induces RNA-protein crosslinks in double-stranded regions of RNA. We show that dsRNA-protein crosslinks can be induced by visible light in the presence of the dye methylene blue, which most likely mediates crosslinking by intercalating in the dsRNA helix. A recombinant dsRNA binding domain from the Drosophila staufen protein and human protein kinase R were crosslinked by UV or methylene blue to a series of dsRNAs. In each case, the degree of crosslinking was greater with methylene blue, particularly with RNAs with few single-stranded loops. Methylene blue-mediated crosslinking therefore complements and extends the existing repertoire of crosslinking methods for detecting RNA-protein interactions.