Non-Coding RNA-Driven Regulation of rRNA Biogenesis

Abstract

Ribosomal RNA (rRNA) biogenesis takes place in the nucleolus, the most prominent condensate of the eukaryotic nucleus. The proper assembly and integrity of the nucleolus reflects the accurate synthesis and processing of rRNAs which in turn, as major components of ribosomes, ensure the uninterrupted flow of the genetic information during translation. Therefore, the abundant production of rRNAs in a precisely functional nucleolus is of outmost importance for the cell viability and requires the concerted action of essential enzymes, associated factors and epigenetic marks. The coordination and regulation of such an elaborate process depends on not only protein factors, but also on numerous regulatory non-coding RNAs (ncRNAs). Herein, we focus on RNA-mediated mechanisms that control the synthesis, processing and modification of rRNAs in mammals. We highlight the significance of regulatory ncRNAs in rRNA biogenesis and the maintenance of the nucleolar morphology, as well as their role in human diseases and as novel druggable molecular targets.

Keywords: non-coding RNAs; nucleolus; rRNA biogenesis; regulatory RNAs.

Figure 1

Illustration of the structure a well-defined nucleolus. The nucleolus consists of three sub-compartments, the fibrillar center (FC), the dense fibrillar center (DFC) and the granular component (GC). FC is surrounded by DFC and both are embedded in GC. At FC borderline the rDNA transcription occurs. The early and late steps of the rRNA processing occur, respectively, at DFC and GC. The early assembly steps occur in GC, where the precursors of 40S (pre-40S) and 60S (pre-60S) subunits are formed, as soon as the 5S rRNA enters the nucleolus. Pol I refers to RNA polymerase I, white and black circles refer to uridylated and methylated modified nucleotides, respectively. Arrows with dashed lines indicate transport from or to the nucleolus.

Figure 2

Examples of negative non-coding RNA regulators of rRNA biogenesis. (Top left panel) pRNA is transcribed by RNA polymerase I (Pol I) by an intergenic spacer promoter (IGS promoter). pRNA is a bifunctional lncRNA which 5′ end forms a DNA-RNA triplex with the T_o element of the rDNA promoter and the methyltransferase DNMT3b which in turn methylates CpG. At its 3′ end, pRNA forms a stem loop structure which interacts with TIP5 component of the nucleolar remodeling complex (NoRC) to induce H3K9me3 histone methylation, removal of H4ac acetylation marks and transcription inhibition by nucleosome shift. (Top right panel) Promoter and pre-antisense lncRNAs (PAPAS) are transcribed by RNA polymerase II (Pol II) from rDNA sequence in an antisense orientation. Under starvation conditions PAPAS interact with the histone methyltransferase Suv4-20h2, leading to trimethylation of histone H4 at lysine 20 (H4K20me3) and chromatin condensation. Under hypoosmotic stress or heat-shock, PAPAS interact with CHD4 and the nucleosome remodeling and deacetylase (NuRD) complex and promote histone deacetylatation, nucleosome shifts that inhibit the rDNA transcription. (Bottom left panel) Long nucleolar RNA (LoNA) is a dual-function lncRNA, transcribed by RNA pol II. LoNA contains two nucleolin-binding sequences, one of which is able to bind and suppress nucleolin’s activity. On the 3′ end, LoNA contains two C/D box sequences that compete for fibrillarin’s binding with canonical C/D box snoRNPs and suppress the pre-rRNA methylation. (Bottom right panel) CircANRIL is a circular lncRNA transcribed by RNA pol II. CircANRIL bears a homology domain with the 47S pre-rRNA which binds PES1, a component of PeBoW complex. Binding of PES1 by circANRIL suppresses PeBoW formation and inhibits ITS2 cleavage. ITS1 refers to Internal transcribed spacer 1 and ITS2 to Internal transcribed spacer 2.

Figure 3

SLERT, a positive regulator of rDNA transcription. SLERT (snoRNA-ended lncRNA enhancing pre-ribosomal RNA transcription) is a snoRNA-ended lncRNA, derived from alternative splicing of TGFB4 pre-mRNA and contains two box H/ACA snoRNAs, the SNORA5C and SNORA5A sequences, at its 5′ and 3′ end, respectively. SLERT enters the nucleolus, binds to DDX21 to alter its conformation, permitting RNA pol I (Pol I) to transcribe the rDNA gene.

Figure 4

aluRNAs role in promoting the rDNA transcription. aluRNAs are transcribed from Alu elements located in intronic regions by RNA pol II (Pol II). aluRNAs are found as separated monomer units (aluRNA_L and aluRNA_R) in cells and could be accumulated in the nucleolus. Under physiological conditions, aluRNAs bind to nucleolin and promote the rDNA transcription. On the other hand, knockdown of aluRNAs causes disruption of the nucleolar structure and reduces the rDNA transcription.

Figure 5

Biogenesis and function of risiRNAs in C. elegans. (1) In response to cellular stress or deficient rRNA modification processing, aberrant rRNAs accumulate and are uridylated at their 3′ end. (2) The uridylated rRNAs are, then, transferred to the cytoplasm, where they are either degraded by 3′-5′ exoribonuclease SUSI-1 (3), or, transcribed by RNA dependent RNA polymerases (RdRPs) to produce risiRNAs (antisense ribosomal siRNAs) (4). risiRNAs can then interact with Argonaute proteins, like NRDE, and are transported to the nucleolus, where they silence the rDNA transcription (5).

Figure 6

Schematic representation of snoRNAs involved in pre-rRNA modification. C/D and H/ACA snoRNPs (small nucleolar ribonucleoproteins) hybridize on pre-rRNA at specific sites that undergo 2′−O methylation and pseudouridylation, respectively. (Lower panel) The C/D box snoRNA contains the conserved C and D motifs where the site-specific 2′-O methylation of the target rRNA is catalyzed by fibrillarin (FBL). In addition, for an active C/D snoRNP, the proteins SNU13 (15.5K), NOP58 and NOP56 are also required. The H/ACA snoRNAs harbor the conserved H and ACA elements that form two pseudouridylation pockets, where pseudouridylation of the target rRNA is catalyzed by dyskerin (DKC1). The core proteins NHP2, NOP10, GAR1 are essential for a functional H/ACA snoRNP.

Figure 7

Schematic representation of SAMMSON regulatory role in pre-rRNA processing. SAMMSON is a lncRNA that is aberrantly expressed in melanoma cells. In the cytoplasm of a melanoma cell, SAMMSON interacts with CARF and then with p32, which is transferred in the mitochondria and promotes the mitochondrial 16S rRNA maturation. Moreover, in the nucleus of a melanoma cell, SAMMSON interacts with XRN2 to induce the snoRNA and pre-rRNA in the nucleolus.