RNA-protein interactions were characterized using the iCLIP protcol (cross-linking and immunoprecipitation). RNA was UV-crosslinked to protein, isolated by immunoprecipitation using mle antibody, and characterized by deep sequencing analysis.

Source was cell extract of clone8 cell line; bait produced from endogenous gene; prey produced from endogenous gene.

Though mle bound to 2,447 sites, roX1 and roX2 sites scored considerably higher, representing over three quarters of all observed cross-linking events.

RNA-protein interactions were characterized by GRNA chromatography. roX1 RNA was tagged with the boxB RNA element, and bound to glutathione beads by virtue of the interaction between boxB and a GST fusion to the λ[[N22]] peptide. This resin was incubated with nuclear extracts, bound proteins were eluted by RNaseA treatment, and characterized by western blot.

Interaction in vitro; bait produced by in vitro transcription; prey derived from wild-type embryonic nuclear extract.

In this assay, roX1 RNA pulled down mle but not msl-2.

RNA bait was tagged with MS2 stem loops, and immobilized on amylose beads using a fusion of MS2-binding protein and maltose binding protein (MBP).

mle binds to a conserved stem loop in roX1 and roX2.

ATP improves the binding affinity and specificity of mle for the conserved roX1/roX2 stem loop.

Interaction in vitro; bait produced by in vitro transcription; prey derived from L2-4 (SL2 cell line derivative) nuclear extract.

Interaction in vitro; bait oligonucleotides synthesized in vitro; prey derived from clone 8 cell extract.

The authors use biotin-tagged oligonucleotides complementary to roX1 to pull down associated factors (protein, RNA or DNA) from cross-linked chromatin.

Factors were detected on polytene chromosomes using antibodies (to proteins) or biotinylated antisense probes (to ncRNA). The PLA signal develops only if probes to the two factors are in close proximity to each other (within tens of nanometers).

Source was larval salivary gland of wild-type fly line; proteins produced from endogenous gene.

Interaction in vitro. Source was cell extract of a clone 8 cell line; bait produced as a recombinant fusion protein in bacterial system; prey produced from endogenous gene.

In presence of ATP, mle significantly enriched lncRNA:roX2 from the S2 transcriptome