6.047/6.878 Lecture 12: Small RNAs Guest Lecture by David Bartel (MIT/Whitehead/HHMI) ([email protected]) Scribed by Boyang Zhao ([email protected]) (2011) September 10, 2012 1 Contents 1 Introduction 3 1.1 ncRNA classifications........................................3 1.2 Small ncRNA.............................................3 1.3 Long ncRNA.............................................3 2 RNA Interference 5 2.1 History of discovery.........................................5 2.2 Biogenesis pathways.........................................5 2.3 Functions and silencing mechanism.................................7 2 6.047/6.878 Lecture 12: Small RNA List of Figures 1 siRNA and miRNA biogenesis pathways..............................6 2 Protein and mRNA changes following miR-223 loss........................7 1 Introduction Large-scale analyses in the 1990s using expressed sequence tags have estimated a total of 35,000 - 100,000 genes encoded by the human genome. However, the complete sequencing of human genome has surprisingly revealed that the numbers of protein-coding genes are likely to be ∼20,000 { 25,000 [12]. While this represents <2% of the total genome sequence, whole genome and transcriptome sequencing and tiling resolution genomic microarrays suggests that over >90% of the genome is still actively transcribed [8], largely as non-protein- coding RNAs (ncRNAs). Although initial speculation has been that these are non-functional transcriptional noise inherent in the transcription machinery, there has been rising evidence suggesting the important role these ncRNAs play in cellular processes and manifestation/progression of diseases. Hence these findings challenged the canonical view of RNA serving only as the intermediate between DNA and protein. 1.1 ncRNA classifications The increasing focus on ncRNA in recent years along with the advancements in sequencing technologies (i.e. Roche 454, Illumina/Solexa, and SOLiD; refer to [16] for a more details on these methods) has led to an explosion in the identification of diverse groups of ncRNAs. Although there has not yet been a consistent nomenclature, ncRNAs can be grouped into two major classes based on transcript size: small ncRNAs (<200 nucleotides) and long ncRNAs (lncRNAs) (≥200 nucleotides) (Table 11)[6,8, 13, 20, 24]. Among these, the role of small ncRNAs microRNA (miRNA) and small interfering RNA (siRNA) in RNA silencing have been the most well-documented in recent history. As such, much of the discussion in the remainder of this chapter will be focused on the roles of these small ncRNAs. But first, we will briefly describe the other diverse set of ncRNAs. 1.2 Small ncRNA For the past decades, there have been a number of well-studied small non-coding RNA species. All of these species are either involved in RNA translation (transfer RNA (tRNA)) or RNA modification and processing (small nucleolar RNA (snoRNA) and small nuclear RNA (snRNA)). In particular, snoRNA (grouped into two broad classes: C/D Box and H/ACA Box, involved in methylation and pseudouridylation, respectively) are localized in the nucleous and participates in rRNA processing and modification. Another group of small ncRNAs are snRNAs that interact with other proteins and with each other to form splicesomes for RNA splicing. Remarkably, these snRNAs are modified (methylation and pseudouridylation) by another set of small ncRNAs - small Cajal body-specific RNAs (scaRNAs), which are similar to snoRNA (in sequence, structure, and function) and are localized in the Cajal body in the nucleus. Yet in another class of small ncRNAs, guide RNAs (gRNAs) have been shown predominately in trypanosomatids to be involved in RNA editing. Many other classes have also been recently proposed (see Table 1) although their functional roles remain to be determined. Perhaps the most widely studied ncRNA in the recent years are microRNAs (miRNAs), involved in gene silencing and responsible to the regulation of more than 60% protein-coding genes [6]. Given the extensive work that has been focused on RNAi and wide range of RNAi-based applications that have emerged in the past years, the next section (RNA Interference) will be entirely devoted to this topic. 1.3 Long ncRNA Long ncRNAs (lncRNAs) make up the largest portion of ncRNAs [6]. However the emphasis placed on the study of long ncRNA has only been realized in the recent years. As a result, the terminology for this family of 1TODO: @scribe: In Table 1, ncRNAs with functions labeled not clear have not yet been extensively searched in literature. There can be recent studies that suggest the functional roles of these ncRNAs. 3 6.047/6.878 Lecture 12: Small RNA Table 1: ncRNA classifications (based on [6,8, 13, 20, 24]) Name Abbreviation Function Housekeeping RNAs Ribosomal RNA rRNA translation Transfer RNA tRNA translation Small nucleolar RNA snoRNA (∼60-220 nt) rRNA modification Small Cajal body-specific RNA scaRNA splicesome modification Small nuclear RNA snRNA (∼60-300 nt) RNA splicing Guide RNA gRNA RNA editing Small ncRNAs (<200 nt) MicroRNA miRNA (∼19-24 nt) RNA silencing Small interfering RNA siRNA (∼21-22 nt) RNA silencing Piwi interacting RNA piRNA (∼26-31 nt) Transposon silencing, epigenetic regulation Tiny transcription initiation RNA tiRNA (∼17-18 nt) Transcriptional regulation? Promoter-associated short RNA PASR (∼22-200 nt) unknown Transcription start site antisense RNA TSSa-RNA (∼20-90 nt) Transcriptional maintainence? Termini-associated short RNA TASR not clear Antisense termini associated short RNA aTASR not clear Retrotransposon-derived RNA RE-RNA not clear 3'UTR-derived RNA uaRNA not clear x-ncRNA x-ncRNA not clear Small NF90-associated RNA snaR not clear Unusually small RNA usRNA not clear Vault RNA vtRNA not clear Human Y RNA hY RNA not clear Long ncRNAs (≥200 nt) Large intergenic ncRNA lincRNA Epigenetics regulation Transcribed ultraconserved regions T-UCR miRNA regulation? Pseudogenes none miRNA regulation? Promoter upstream transcripts PROMPT Transcriptional activation? Telomeric repeat-containing RNA TERRA telomeric heterochromatin main- tenance GAA-repeat containing RNA GRC-RNA not clear Enhancer RNA eRNA not clear Long intronic ncRNA none not clear Antisense RNA aRNA not clear Promoter-associated long RNA PALR not clear Stable excised intron RNA none not clear Long stress-induced non-coding transcripts LSINCT not clear ncRNAs are still in its infancy and oftentimes inconsistent in the literature. This is also in part complicated by cases where some lncRNAs can also serve as transcripts for the generation of short RNAs. In light of these confusions, as discussed in the previous chapter, lncRNA have been arbitrarily defined as ncRNAs with size greater than 200 nts (based on the cut-off in RNA purification protocols) and can be broadly categorized into: sense, antisense, bidirectional, intronic, or intergenic [19]. For example, one particular class of lncRNA called long intergenic ncRNA (lincRNA) are found exclusively in the intergenic region and possesses chromatin modifications indicative of active transcription (e.g. H3K4me3 at the transcriptional 4 6.047/6.878 Lecture 12: Small RNA start site and H3K36me3 throughout the gene region) [8]. Despite the recent rise of interest in lncRNAs, the discovery of the first lncRNAs (XIST and H19 ), based on searching cDNA libraries, dated back to the 1980s and 1990s before the discovery of miRNAs [3,4]. Later studies demonstrated the association of lncRNAs with polycomb group proteins, suggesting potential roles of lncRNAs in epigenetic gene silencing/activation [19]. Another lncRNA, HOX Antisense Intergenic RNA (HOTAIR), was recently found to be highly upregulated in metastatic breast tumors [11]. The association of HOTAIR with the polycomb complex again supports a potential unified role of lncRNAs in chromatin remodeling/epigenetic regulation (in either a cis-regulatory (XIST and H19 ), or trans-regulatory (e.g. HOTAIR) fashion) and disease etiology. Recent studies have also identified HULC and pseudogene (transcript resembling real genes but contains mutations that prevent their translation into functional proteins) PTENP1 that may function as a decoy in binding to miRNAs to reduce the overall effectiveness of miRNAs [18, 25]. Other potential roles of lncRNAs remains to be explored. Nevertheless, it is becoming clear that lncRNAs are less likely to be the result of transcriptional noise, but may rather serve critical role in the control of cellular processes. 2 RNA Interference RNA interference has been one of the most significant and exciting discoveries in recent history. The impact of this discovery is enormous with applications ranging from knockdown and loss-of-function studies to the generation of better animal models with conditional knockdown of desired gene(s) to large-scale RNAi-based screens to aid drug discovery. 2.1 History of discovery The discovery of the gene silencing phenomenon dated back as early as the 1990s with Napoli and Jorgensen demonstrating the down-regulation of chalcone synthase following introduction of exogenous transgene in plants [17]. Similar suppression was subsequently observed in other systems [10, 22]. In another set unrelated work at the time, Lee et al. identified in a genetic screen that endogenous lin-4 expressed a non-protein- coding product that is complementary to the lin-14 gene and controlled the timing of larval development (from first to second larval state) in C. elegans [15]. We now know this as the first miRNA to be discovered. In 2000, another miRNA, let-7, was discovered in the same