UPA-seq: prediction of functional lncRNAs using differential sensitivity to UV crosslinking

Abstract

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, we 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. We propose that UPA-seq provides a useful tool for the selection of lncRNA candidates to be analyzed in depth in subsequent functional studies.

Molecular dissection of nuclear paraspeckles: towards understanding the emerging world of the RNP milieu

“…A group of novel lncRNAs have been identified according to their semi-extractable properties, and many of them localized to distinct foci in the nucleus, probably representing novel RNP milieux [96]. Another unique property of NEAT1 is that it is easily cross-linked to associating proteins upon UV-irradiation [97] and differential sensitivity to the UV-induced cross-linking further identified a larger group of lncRNAs, some of which also form a cloud of RNA at the putative transcription sites [97] (figure 4b,c). Interestingly, treatment with 1,6-hexanediol, which disrupts amphipathic interaction of IDPs, improved the extraction of semi-extractable lncRNAs and decreased sensitivities to UV-cross-linking, suggesting that they are bona fide components of the phase-separated RNP milieu ( [97]; T Chujo, T Hirose 2017, unpublished observation).…”

“…Another unique property of NEAT1 is that it is easily cross-linked to associating proteins upon UV-irradiation [97] and differential sensitivity to the UV-induced cross-linking further identified a larger group of lncRNAs, some of which also form a cloud of RNA at the putative transcription sites [97] (figure 4b,c). Interestingly, treatment with 1,6-hexanediol, which disrupts amphipathic interaction of IDPs, improved the extraction of semi-extractable lncRNAs and decreased sensitivities to UV-cross-linking, suggesting that they are bona fide components of the phase-separated RNP milieu ( [97]; T Chujo, T Hirose 2017, unpublished observation). mRNAs that localize to P-bodies and neuronal granules also exhibit increased sensitivity to UV-cross-linking to proteins [97], suggesting that this is a general feature of RNA components in the RNP milieu.…”

“…Interestingly, treatment with 1,6-hexanediol, which disrupts amphipathic interaction of IDPs, improved the extraction of semi-extractable lncRNAs and decreased sensitivities to UV-cross-linking, suggesting that they are bona fide components of the phase-separated RNP milieu ( [97]; T Chujo, T Hirose 2017, unpublished observation). mRNAs that localize to P-bodies and neuronal granules also exhibit increased sensitivity to UV-cross-linking to proteins [97], suggesting that this is a general feature of RNA components in the RNP milieu.…”

Forced isoform switching of Neat1_1 to Neat1_2 leads to the loss of Neat1_1 and the hyperformation of paraspeckles but does not affect the development and growth of mice

“…Paraspeckles are phase-separated nonmembranous nuclear bodies that are unique to mammalian species (Fox et al 2018;Nakagawa et al 2018). Neat1 tightly associate with proteins (Ishizuka et al 2014;Komatsu et al 2018); more than 40 proteins assemble at a specific region of Neat1 (Fox et al 2002(Fox et al , 2005Dettwiler et al 2004;Naganuma et al 2012;Fong et al 2013;Hu et al 2015;Kawaguchi et al 2015) and form a characteristic core-shell structure (Souquere et al 2010;West et al 2016). Paraspeckles have been proposed to function via the sequestration of paraspeckle-associated RNA binding proteins within these nuclear bodies (Hirose et al 2014;Imamura et al 2014).…”