Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Endogenous siRNAs and noncoding RNA-derived small RNAs are expressed in adult mouse hippocampus and are up-regulated in olfactory discrimination training NEIL R. SMALHEISER,1 GIOVANNI LUGLI,1 JYOTHI THIMMAPURAM,2 EDWIN H. COOK,1 and JOHN LARSON1 1Department of Psychiatry, University of Illinois at Chicago, Chicago, Illinois 60612, USA 2W.M. Keck Center for Comparative and Functional Genomics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA ABSTRACT We previously proposed that endogenous siRNAs may regulate synaptic plasticity and long-term gene expression in the mammalian brain. Here, a hippocampal-dependent task was employed in which adult mice were trained to execute a nose-poke in a port containing one of two simultaneously present odors in order to obtain a reward. Mice demonstrating olfactory discrimination training were compared to pseudo-training and nose-poke control groups; size-selected hippocampal RNA was subjected to Illumina deep sequencing. Sequences that aligned uniquely and exactly to the genome without uncertain nucleotide assignments, within exons or introns of MGI annotated genes, were examined further. The data confirm that small RNAs having features of endogenous siRNAs are expressed in brain; that many of them derive from genes that regulate synaptic plasticity (and have been implicated in neuropsychiatric diseases); and that hairpin-derived endo-siRNAs and the 20- to 23-nt size class of small RNAs show a significant increase during an early stage of training. The most abundant putative siRNAs arose from an intronic inverted repeat within the SynGAP1 locus; this inverted repeat was a substrate for dicer in vitro, and SynGAP1 siRNA was specifically associated with Argonaute proteins in vivo. Unexpectedly, a dramatic increase with training (more than 100-fold) was observed for a class of 25- to 30-nt small RNAs derived from specific sites within snoRNAs and abundant noncoding RNAs (Y1 RNA, RNA component of mitochondrial RNAse P, 28S rRNA, and 18S rRNA). Further studies are warranted to characterize the role(s) played by endogenous siRNAs and noncoding RNA-derived small RNAs in learning and memory. Keywords: RNA interference; synaptic plasticity; learning; Lrrtm1; SynGAP1; Dgcr8; dicer; snoRNAs INTRODUCTION so-called RISC protein complex that binds to complemen- tary mRNAs; if the siRNA is a perfect match to its target, RNA interference (RNAi) pathways are ubiquitous through- the target mRNA is cleaved and thus rapidly destroyed. out the animal (and plant) kingdoms and comprise two main We have proposed that RNA interference has features that pathways—one that generates siRNAs from sense-antisense theoretically make it an attractive mechanism for regulating hybrid transcripts or other types of double-stranded RNA synaptic plasticity and other long-term changes in gene ex- (dsRNA) structures (including inverted repeats or ‘‘hair- pression in the mammalian brain, and predicted that endog- pins’’), and one that generates microRNAs from small stem– enous siRNAs (endo-siRNAs) should be formed within loop pre-miRNA precursors (for review, see Carthew and neurons at the onset of learning (Smalheiser et al. 2001). Sontheimer 2009). The siRNA pathway generates z22-nt However, early cloning studies in numerous species failed to dsRNAs, of which one strand is incorporated into the detect endo-siRNAs against mRNA target sequences. With the advent of deep sequencing technologies, it is now appre- ciated that endo-siRNAs complementary to mRNAs are ex- Abbreviations: siRNA, small inhibitory RNA; miRNA, microRNA; endo- siRNA, endogenous siRNA; RISC, RNA induced silencing complex. pressed in Caenorhabditis elegans and Drosophila as well as Reprint requests to: Neil R. Smalheiser, Department of Psychiatry, UIC, mouse oocytes (fore reviews, see Okamura and Lai 2008; MC912, 1601 W. Taylor St., Chicago, IL 60612, USA; e-mail: [email protected]; Ghildiyal and Zamore 2009). Endo-siRNAs against mRNA fax: (312) 413-4569. Article published online ahead of print. Article and publication date are sequences are expressed in ES cells (Babiarz et al. 2008), in at http://www.rnajournal.org/cgi/doi/10.1261/rna.2123811. HepG2 liver carcinoma cells (Kawaji et al. 2008), and in 166 RNA (2011), 17:166–181. Published by Cold Spring Harbor Laboratory Press. Copyright Ó 2011 RNA Society. Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Expression of novel small RNAs in hippocampus developing skin as well (Yi et al. 2009). However, apart from under consideration here, a total of 65,516 unique RNA two reports regarding the vertebrate slc34a gene (Carlile et al. sequences mapped uniquely and exactly to 14,583 known 2008, 2009), there is still virtually no evidence that endo- genes (Supplemental File 1). siRNAs play dynamic roles in any physiological process. The resulting small RNA sequences exhibited a sharp peak In order to test whether small RNAs having the features in abundance at the 21- to 22-nt size class (Fig. 1). The vast of endo-siRNAs are expressed in the brain and whether majority of these can be considered candidates to represent they are modulated during the learning process, we em- endo-siRNAs, given that (1) the deep sequencing method ployed a hippocampal-dependent task in which adult mice was designed to amplify RNAse III cleavage products selec- were trained to execute a nose-poke in a port containing one tively (since adaptors were added selectively to RNAs that of two simultaneously-present odors in order to obtain a have a free –OH group on the 39-end and a monophosphate reward (Larson and Sieprawska 2002). Mice demonstrating on the 59-end), (2) we excluded sequences that map to known discrimination learning were compared to two yoked control miRNA genes, and (3) the sequences all mapped uniquely to groups: (1) mice that were exposed to the same two odors exons or introns of MGI genes, most of which encode but reward was not contingent upon discriminative respond- protein-coding mRNAs. Indeed, as shown below, several of ing (pseudo-training), and (2) mice that were not exposed to the putative endo-siRNAs exhibited characteristic features odor pairs at all (nose-poke). In all groups, mice performed strongly suggesting that they arise from dicer processing of the same number of trials, with the same motor responses RNA hairpin-like inverted repeats or sense-antisense tran- (nose-pokes). Any changes observed in the training vs. script hybrids. pseudo-training comparison are likely to reflect the process In contrast, the data set appears to be depleted of other of learning to associate a specific odor with reward. We chose known small RNAs such as random mRNA degradation 70% responses correct as a criterion for learning because that products (these would not be expected to produce a tight is the earliest point at which mice behave significantly dif- peak at 21-22 nt or to be acceptors for adaptors during deep ferent from chance at P = 0.05. The training group consisted sequencing), TSSa-RNAs (these should comprise overlap- of seven mice that reached criterion after three sessions of 20 ping sense and antisense positions near the transcriptional trials (requiring z40 min of training); each trained mouse start site, generally upstream of the mRNA transcript) (Seila was yoked to a pseudo-trained mouse and a mouse simply et al. 2008), piRNAs (these do not appear to be expressed in performing nose-pokes. Thus, the experimental design ex- brain, are not RNAse III products, are >23 nt, often map to amines changes that occur near the onset of the learning genomic repeats, and sometimes form overlapping pairs process. with a 10-nt offset), or tRNA-derived cleavage products. We have previously characterized miRNA expression in these mice and found that training up-regulated miRNA levels and reorganized miRNA co-expression modules Putative endo-siRNAs that map to predicted hairpin (Smalheiser et al. 2010). In the present study, total RNA RNA inverted repeats was pooled (three or four mice per pool, giving two pooled To begin examining the data set in detail, we looked for samples per treatment group), and Illumina deep sequencing sequences whose features suggested that they derived from of size-selected small RNAs was carried out. We report that hairpin inverted repeats. Each genomic locus in the data set endo-siRNAs and other ncRNA-derived RNAs are robustly was examined to identify cases in which multiple small expressed in the adult mouse hippocampus, some of which RNAs mapped to closely adjacent sites in sense orienta- showed surprisingly large changes that were specific to the tion, to regions characteristic of RNA hairpins or linear training group. RESULTS Using the Illumina system, each pooled sample provided 12– 13 million raw sequence reads or ‘‘counts’’ (Supplemental Table S1). Here, we consider the data set of small RNAs that aligned (‘‘mapped’’) exactly and uniquely to the reference mouse genome within exons and introns of annotated MGI (Mouse Genome Informatics) gene entries. Excluded were sequences that contained uncertain nucleotide assignments or that mapped to more than one locus, as well as those that mapped to annotated miRNA pre-miR sequences found in FIGURE 1. Length distribution of small RNAs in the data set. Shown miRBase. The number of ‘‘raw’’ sequence counts for each are the total number of sequence reads, and the total number of unique sequences, observed in the entire data set (across all samples unique sequence (filtered and cleansed but non-normalized) and treatment groups). A more detailed breakdown of the data set is was tabulated for each sample. Across the filtered data set presented in Table 1. www.rnajournal.org 167 Downloaded from rnajournal.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Smalheiser et al.