The long noncoding RNA SPRIGHTLY acts as an intra-nuclear organizing hub for pre-mRNA molecules.. Homo sapiens

We report the application of domain-specific chromatin isolation by RNA purification (dChIRP) to identify RNA binding partners of long coding RNA, SPRIGHTLY. We demonstrate by SHAPE-Seq and dChIRP that SPRIGHTLY RNA secondary structure has a core pseudo-knotted domain. This lncRNA interacts with the intronic regions of six pre-mRNAs: SOX5, SMYD3, SND1, MEOX2, DCTN6, and RASAL2, all of which have cancer related functions. Hemizygous knockout of SPRIGHTLY by CRISPR/Cas9 in melanoma cells significantly decreases SPRIGHTLY lncRNA levels, simultaneously decreases the levels of its interacting pre-mRNA molecules, decreases anchorage-independent growth rate of cells, and the rate of in vivo tumor growth in mouse xenografts. These results provide the first demonstration of a lncRNA’s 3-dimensional coordinating role in facilitating cancer-related gene expression in human melanomas. Overall design: Three sets of probes corresponding to three regions of SPRIGHTLY were desiged to pull-down target RNA SPRIGHTLY. Melanoma cell line, A375, cells were treated with 0.3% formaldehde. Chromatin were prepared and subjected to pull-down with three sets of probes separately. Pull-downed RNAs were extracted and subjected to RNA-sequencing using a HiSeq 2500 (Illumina).