Contents lists available at ScienceDirect Journal of Proteomics journal homepage: www.elsevier.com/locate/jprot Functional characterization of RNA fragments using high-throughput interactome screening Paulina Jackowiak a, ⁎ , Angelika Lis a , Magdalena Luczak a,b , Ireneusz Stolarek a , Marek Figlerowicz a,c, ⁎ a Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland b Institute of Chemical Technology and Engineering, Poznan University of Technology, ul. Berdychowo 4, 60-965 Poznan, Poland c Institute of Computing Science, Poznan University of Technology, Piotrowo 3A, 60-965 Poznan, Poland ARTICLE INFO Keywords: RNA fragments Protein interactome RNA-protein interaction ABSTRACT Populations of small eukaryotic RNAs, in addition to relatively well recognized molecules such as miRNAs or siRNAs, also contain fragments derived from all classes of constitutively expressed non-coding RNAs. It has been recently demonstrated that the formation and accumulation of RNA fragments (RFs) is cell-/tissue-specifc and depends on internal and external stimuli. Unfortunately, the mechanisms underlying RF biogenesis and function remain unclear. To better understand them, we employed RNA pull-down and mass spectrometry methods to characterize the interactions of seven RFs originating from tRNA, snoRNA and snRNA. By integrating our results with publicly available data on physical protein-protein interactions, we constructed an RF interactome network. We determined that the RF interactome comprises proteins generally diferent from those that interact with their parental full length RNAs. Proteins captured by the RFs were involved in mRNA splicing, tRNA processing, DNA recombination/replication, protein biosynthesis and carboxylic acid metabolism. Our data suggest that RFs can be endogenous aptamer-like molecules and potential players in recently revealed RNA-protein regulatory net- works. Significance: In the recent decade it has become evident that RNAs with well-known functions (for example tRNA, snoRNA or rRNA) can be cleaved to yield short fragments, whose role in cells remains only partially characterized. At the same time, unconventional interactions between mRNA and proteins without RNA-binding domains have been demonstrated, revealing novel layers of possible RNA-mediated regulation. Considering the above, we hypothesized that RNA fragments (RFs) can be endogenous aptamer-like molecules that un- conventionally interact with proteins. In this study we identifed protein partners of seven selected RFs. We found that RFs bind diferent set of proteins than their parental full length RNAs and identifed proteins dif- ferentially bound by the particular RFs. These observations suggest biological relevance of the discovered in- teractions. Our data provide a novel perspective on the signifcance of RFs and point to this pool of molecules as to a rich collection of potential components of the recently discovered RNA-protein regulatory networks. 1. Introduction Small RNA fragments (RFs) derived from multiple RNA classes are important components of plant, animal and human transcriptomes. For some RFs, their involvement in various biological processes has been shown [1–4]. Even though the spectrum of recognized RF functions has been expanding [5–10], a wealth of diverse RFs identifed across spe- cies suggests that their actual signifcance is much greater than ori- ginally anticipated. In addition, the biogenesis of RFs is still poorly understood as the nucleases implicated in RF generation have been identifed in only a few cases [11–14]. Recently, we characterized the RF repertoire of a hepatitis C virus cell culture model (HCVcc) [15]. Analysis of RNA cleavage patterns revealed that RFs often include the protein binding motifs present in their parental molecules. This ob- servation raises questions about two points: (i) the signifcance of protein shields in RF biogenesis and (ii) the possibility of RF-mediated regulation of cellular pathways by either sequestering specifc protein factors or organizing proteins in functional complexes. Without a doubt, the frst step towards answering these questions is to identify the pro- tein partners of RFs. Recently, two complementary methodological approaches have been developed to study native ribonucleoprotein (RNP) complexes. On https://doi.org/10.1016/j.jprot.2018.10.007 Received 16 February 2018; Received in revised form 17 August 2018; Accepted 15 October 2018 ⁎ Corresponding authors at: Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland. E-mail addresses: paulinaj@ibch.poznan.pl (P. Jackowiak), marekf@ibch.poznan.pl (M. Figlerowicz). Journal of Proteomics xxx (xxxx) xxx–xxx 1874-3919/ © 2018 Published by Elsevier B.V. Please cite this article as: Jackowiak, P., Journal of Proteomics, https://doi.org/10.1016/j.jprot.2018.10.007