UPA-Seq – prediction of functional lncRNAs using differential sensitivity to UV crosslinking

While a large number of long noncoding RNAs (lncRNAs) are transcribed from the genome of higher eukaryotes, systematic prediction of their functionality has been challenging due to the lack of conserved sequence motifs or structures. Assuming that some lncRNAs function as large ribonucleoprotein complexes and thus are easily crosslinked to proteins upon UV irradiation, Hokkaido University researchers performed RNA-Seq analyses of RNAs recovered from the aqueous phase after UV irradiation and phenol-chloroform extraction (UPA-Seq). As expected, the numbers of UPA-Seq reads mapped to known functional lncRNAs were remarkably reduced upon UV irradiation. Comparison with ENCODE eCLIP data revealed that lncRNAs that exhibited greater decreases upon UV irradiation preferentially associated with proteins containing prion-like domains (PrLDs). Fluorescent in situ hybridization (FISH) analyses revealed the nuclear localization of novel functional lncRNA candidates, including one that accumulated at the site of transcription. The researchers propose that UPA-Seq provides a useful tool for the selection of lncRNA candidates to be analyzed in depth in subsequent functional studies.

Identification of novel functional lncRNA candidates by UPA-Seq

(A) MA-plot of log2 fold change of reads upon UV irradiation in HippCulture andHepG2 cells as a function of the relative abundance (log10 baseMeans). Light green dotsrepresent lncRNAs that exhibited significant (FDR < 0.05) fold change, orange dotsrepresent functional lncRNA candidates used for the following FISH studies, and darkgreen dots represent known functional lncRNAs. Note that only lncRNAs with negativelog2 fold change values are illustrated. (B) Group of functional lncRNA candidates thatexhibited significant decrease (FDR < 0.05) upon UV irradiation. Genes are categorizedinto known genes (annotated) and unknown genes assigned with ID numbers withvarious prefixes (RP, Gm, AC, and AL). (C, D) FISH images showing the subcellulardistribution of functional lncRNA candidates (green) in HippCulture cells (C) andHepG2 cells (D). Magenta denotes nuclei counterstained with DAPI. Scale bar, 10 μm. (E) Schematic illustration showing the gene organization at the Rab30/RP11-113K21.5locus and the positions of the probes used for simultaneous detection. Note that thesignals obtained with probes that detect RP11-113K21.5 were closely associated withthe signals obtained with probes that detect Rab30 intron sequences. Dashed linesrepresent the position of the nucleus. Scale Bar, 10 μm. (F) Half-lives of 18S rRNA,MALAT1, NEAT1, RP11-113K21.5, RP11-46C20.1, and SNHG1, as measured byBRIC RT-qPCR. Note that the functional lncRNA candidates exhibited relatively short half-lives.