Functional dissection of NEAT1 using genome editing reveals substantial localization of the NEAT1_1 isoform outside paraspeckles.
Large numbers of long noncoding RNAs have been discovered in recent years, but only a few have been characterized. NEAT1 (nuclear paraspeckle assembly transcript 1) is a mammalian long noncoding RNA that is important for the reproductive physiology of mice, cancer development, and the formation of subnuclear bodies termed paraspeckles. The two major isoforms of NEAT1 (3.7 kb NEAT1_1 and 23 kb NEAT1_2 in human) are generated from a common promoter and are produced through the use of alternative transcription termination sites. This gene structure has made the functional relationship between the two isoforms difficult to dissect. Here we used CRISPR-Cas9 genome editing to create several different cell lines: total NEAT1 knockout cells, cells that only express the short form NEAT1_1, and cells with twofold more NEAT1_2. Using these reagents, we obtained evidence that NEAT1_1 is not a major component of paraspeckles. In addition, our data suggest NEAT1_1 localizes in numerous nonparaspeckle foci we termed "microspeckles," which may carry paraspeckle-independent functions. This study highlights the complexity of lncRNA and showcases how genome editing tools are useful in dissecting the structural and functional roles of overlapping transcripts.
Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Nedlands, Western Australia 6009, Australia.|School of Human Sciences, The University of Western Australia, Crawley, Western Australia 6009, Australia.
Ruohan
R
Li
School of Human Sciences, The University of Western Australia, Crawley, Western Australia 6009, Australia.|Western Australian Neuroscience Research Institute, Nedlands, Western Australia 6009, Australia.
Alan R
AR
Harvey
School of Human Sciences, The University of Western Australia, Crawley, Western Australia 6009, Australia.|Western Australian Neuroscience Research Institute, Nedlands, Western Australia 6009, Australia.
Stuart I
SI
Hodgetts
Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Nedlands, Western Australia 6009, Australia archa.fox@uwa.edu.au.|School of Human Sciences, The University of Western Australia, Crawley, Western Australia 6009, Australia.
Archa H
0000-0003-1962-270X
AH
Fox
D064113:CRISPR-Cas Systems; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D000072669:Gene Editing; D055786:Gene Knockout Techniques; D006801:Humans; D059368:RNA Isoforms; D000072260:RNA Splicing Factors / Q000378:metabolism; D034443:RNA Transport; D000094704:RNA, Guide, CRISPR-Cas Systems; D062085:RNA, Long Noncoding / Q000235:genetics*
C577276:NEAT1 long non-coding RNA, human; D059368:RNA Isoforms; D000072260:RNA Splicing Factors; D000094704:RNA, Guide, CRISPR-Cas Systems; D062085:RNA, Long Noncoding
CRISPR genome editing; DBHS proteins; NEAT1; long noncoding RNAs; paraspeckles