RNA Metabolism and the Role of Small RNAs in Regulating Multiple Aspects of RNA Metabolism

Abstract

RNA metabolism is focused on RNA molecules and encompasses all the crucial processes an RNA molecule may or will undergo throughout its life cycle. It is an essential cellular process that allows all cells to function effectively. The transcriptomic landscape of a cell is shaped by the processes such as RNA biosynthesis, maturation (RNA processing, folding, and modification), intra- and inter-cellular transport, transcriptional and post-transcriptional regulation, modification, catabolic decay, and retrograde signaling, all of which are interconnected and are essential for cellular RNA homeostasis. In eukaryotes, sRNAs, typically 20-31 nucleotides in length, are a class of ncRNAs found to function as nodes in various gene regulatory networks. sRNAs are known to play significant roles in regulating RNA population at the transcriptional, post-transcriptional, and translational levels. Along with sRNAs, such as miRNAs, siRNAs, and piRNAs, new categories of ncRNAs, i.e., lncRNAs and circRNAs, also contribute to RNA metabolism regulation in eukaryotes. In plants, various genetic screens have demonstrated that sRNA biogenesis mutants, as well as RNA metabolism pathway mutants, exhibit similar growth and development defects, misregulated primary and secondary metabolism, as well as impaired stress response. In addition, sRNAs are both the "products" and the "regulators" in broad RNA metabolism networks; gene regulatory networks involving sRNAs form autoregulatory loops that affect the expression of both sRNA and the respective target. This review examines the interconnected aspects of RNA metabolism with sRNA regulatory pathways in plants. It also explores the potential conservation of these pathways across different kingdoms, particularly in plants and animals. Additionally, the review highlights how cellular RNA homeostasis directly impacts adaptive responses to environmental changes as well as different developmental aspects in plants.

Keywords: PTGS; RNA editing; RNA modification; RNA processing; Retrograde signaling; TGS; Transcription; mRNA; mRNA decay; non-coding RNAs; small RNAs.

Figure 1

sRNA biogenesis models for plants and animals. (A) miRNA biogenesis pathway—RNAPII transcribes the MIR specific. Primary miRNA (pri-miRNA) is then spliced into a single hairpin structure by DICER-LIKE 1, which furthermore gets cleaved into a miRNA duplex of 21 nt with the help of nuclear dicing bodies (D-bodies) including DICER-LIKE 1/3/4 (DCL1/3/4), HYPONASTIC LEAVES 1 (HYL1), SERRATE (SE), and TOUGH (TGH). The duplex is then methylated at the 3′ ends catalyzed with HUA ENHANCER1. With the assistance of HEAT SHOCK PROTEIN70/90 (HSP70/90), the mature miRNA gets loaded into the ARGONAUTE1/10 (AGO1/10) (also known as RISC) based on the specific miRNAs. (B) Secondary siRNAs (phasiRNA, tasiRNA, and easiRNA) biogenesis model—After PHAS loci, TAS loci, and active transposons are transcribed via RNAP II, AGO1/7-loaded mature miRNA cleaves the transcribed mRNA. 5′ fragment of the cleaved mRNA degrades, while the 3′ strand is converted into a double strand with the help of RDR6. SGS3 and SDE5 help in recruiting RDR6 to the recognition site. DCL4/3 participates in ta-siRNA and phasiRNA production, whereas DCL2/4 participates in easiRNA production. The 21–24 nt mature siRNA strand then gets loaded into AGO1/7 for downstream gene regulation. (C) Plant endogenous siRNA biogenesis—The transposable elements, repetitive regions, or gene introns get transcribed with RNAP IV, which then gets converted into dsRNA with the help of RDR2/4. The dsRNA then gets cleaved into 20–24 nt fragments with the help of DCL2/3/4. HSP90 then helps to load the mature siRNA strand in the respective AGO4/6/9, based on their origin. (D) Canonical and miRNA Biogenesis in Animals—After the transcription of the MIR-specific locus with DNA-dependent RNA polymerase II, pri-miRNAs were converted to single hairpin-like structures (pre-miRNAs) with the help of Drosha. The pre-miRNA transported into the cytoplasm with Exportin1/5 proteins then gets further cleaved into miRNA duplexes by Dicer proteins. The pathways create this miRNA duplex without Dorsal/Dicer acting upon the primary miRNA. The mature miRNA strand then gets loaded in the AGO2. (E) Endo- and exogenous modes of siRNA production in Caenorhabditis elegans—siRNAs derived from ssRNA, and dsRNA are loaded into primary Argonaute proteins, ERGO-1 and RDE1, respectively. The loaded primary Argonaute protein, with the help of RRF1 and Mutator, mediates the conversion of 26 nt long 5′-guanosine siRNA into 22G siRNA. The produced siRNAs are then loaded into the secondary Argonaute proteins for downstream gene silencing. (F) Biogenesis of piRNA and the regulatory ping pong cycle of biogenesis in Drosophila melanogaster—piRNA gene sequences are marked by an upstream Ruby motif. The piRNA precursors are transcribed by RNAP II and then exported to the cytoplasm. These precursors are then processed by endonuclease Zucchini and an unknown 3′–5′ exonuclease. Via DmHen1/Pimet methyltransferase, the 3′ end of the mature piRNA gets 2′-O-methylated. The mature piRNA gets loaded into PIWI, forming piRISC to regulate methylation of TEs. Apart from PIWI protein alone, some gets loaded into Aub, which then initiates the ping pong cycle of biogenesis. Aub loaded with piRNA and AGO3 loaded with secondary piRNA repress TE activity through DNA cytosine methylation. PIWI-related Gene 1 (PRG-1) is required for primary piRNA activity, whereas HRDE-1 (Heritable RNA interference (RNAi) deficient protein-1) is the Argonaute protein that carries RdRP-amplified 22 nt, 5′-guanosine siRNA (22-GsiRNA). (Modified from [16]).

Figure 2

PTGS and TGS Mode of Action for miRNA, siRNA, and piRNA in Plants and Animals. (A,B) Post-transcriptional gene silencing of plant and animal miRNAs through mRNA cleavage, RNA decay, and translational repression. (C) Exogenous siRNA is only able to participate in PTGS through mRNA cleavage. (D) Plant and animals endogenous siRNA and piRNAs, the loaded AGO protein, after being transported back to the nucleus, target nascent RNA Pol-V transcripts (line represented in red) through complementary siRNA and form the RdDM complex (RNA-dependent DNA methylation). GW/WG protein, associated with RNAP V, KTF1 acts as an organizer by coordinating with AGO and 5-meC (5-methylCytosine). Similarly, the AGO-associated protein RDM1 interacts with DRM2, a RdDM complex catalytically active de novo methyltransferase, and binds with single-stranded methylated DNA. DRM3, a catalytically inactive paralogue of DRM2, is also known to be involved in the RdDM complex, but its function is still unknown. After all these proteins are localized, DRM2 catalyzes methylation of cytosine in all sequence contexts. (Modified from [16,18]).

Figure 3

miRNA-mediated mRNA decay pathway in plants and animals. (A) miRNA binds to the complementary site in the Open Reading frame and induces endonucleolytic cleavage at the splice site (between the nucleotides 10 and 11). The 5′ fragment is uridylated by HUA Enhancer 1 Suppressor 1 (HESO 1) and is degraded by XRN4 in a 5′ to 3′ direction. Similarly, the 3′ cleaved fragments are degraded by XRN4 without uridylation. (B) miRNA, after attaching with the activated mRNA, recruits CCR4-NOT and PAN2-PAN3 deadenylase complexes to target mRNAs via the GW182 protein. These deadenylated mRNAs are then oligouridylated by TUT4/7, thus starting the general mRNA decay in mammals. Apart from deadenylation, GW182 can also promote dissociation of PAPB (poly(A) binding protein). DDX6 (the de-capping activators) are then recruited onto the CCR4-NOT complex. This helps the DCP2 enzyme in removing the 5′ 7-methylated guanine cap. Finally, XRN1 acts on the uncapped uridylated mRNA strand by performing 5′-3′ exonucleolytic decay. (Modified from [127]).