Poster abstracts

Poster number 49 submitted by Lalith Gunawardane

Analysis of the “interactome” of a long non-coding RNA: lncRNA-Protein Interactions Capture by AntiSense Oligonucleotides (lncRNA-PICASO)

Lalith S. Gunawardane (Department of Biochemistry, Case Western Reserve University ), Bing Zhang (Department of Biochemistry, Case Western Reserve University ), Hiroto Kambara (Department of Biochemistry, Case Western Reserve University ), Saba Valadkhan (Department of Biochemistry, Case Western Reserve University )

Abstract:
While the long non-coding RNAs constitute a large portion of the mammalian transcriptome, the mechanistic aspects of their biological functions have remained mostly elusive. To gain insight into the relationship between the function of a long non-coding RNA and the complement of cellular factors with which it interacts, we have developed an in vivo pull down approach. This method is based on the use of biotinylated antisense oligos (ASOs) to pull down a targeted RNA and its associated proteins from cellular extracts. To ensure that the observed RNA-protein interactions reflect those occurring in vivo, cells are crosslinked prior to preparation of extracts. In order to distinguish the bona-fide “interactome” proteins that copurify with the target RNA from those that are present in the pull down fraction due to possible off-target interactions of ASOs, at least six different ASOs will be used. These will include four non-targeting ASOs and at least two ASOs that are complementary to different sequences in the target RNA and will be individually used in six parallel pull down reactions. The co-purified proteins will be identified by mass spectrometry and those that are co-purified with both targeting ASOs but are not found in pull down fractions obtained with non-targeting ASOs, will be identified as part of the interactome of the targeted RNA. We have successfully employed this method on BORG lncRNA and have identified ~ 20 proteins which specifically and reproducibly co-purified with BORG. We have confirmed the mass spectrometry results by western blot, and importantly, have been able to validate the interactions captured by lncRNA-PICASO by reciprocal immunoprecipitation of the identified proteins followed by RT-PCR for BORG (RNA IP). This method permits the purification of endogenous lncRNAs without a need for addition of sequence elements such as MS2 loops, which may interfere with biogenesis or function of lncRNAs. Our results suggest that lncRNA-PICASO is a robust method for defining the interactome of both lncRNAs and mRNAs. Further, the described method is sensitive enough for purification of relatively low abundance transcripts, which makes it applicable to a wide range of cellular long non-coding transcripts.

Keywords: long non-coding RNA, interactome