pre-rRNA Processing Research Articles

YTHDF2 contributes to psoriasis by promoting proliferation and inflammatory response through regulation of the Wnt signaling pathway

YT521-B homology domain family 2 (YTHDF2), a pivotal m6A-binding protein, is now understood to significantly influence a diverse array of biological functions, including cell migration, proliferation, differentiation, and inflammatory responses. Additionally, YTHDF2 participates in mRNA decay and pre-rRNA processing. This study explored the specific role of YTHDF2 in the pathogenesis of psoriasis and its underlying mechanisms. Our preliminary findings revealed upregulation of YTHDF2 expression in psoriasis. Subsequent silencing of YTHDF2 in a psoriatic cell model resulted in a marked decrease in mRNA expression of IL-17A, S100A8, and S100A9, accompanied by a reduction in cell proliferation. Conversely, overexpression of YTHDF2 led to the opposite effects. Treatment with DC-Y27-13, a YTHDF2 inhibitor, demonstrated a therapeutic effect in psoriasis mice. Next, mRNA sequencing analysis identified significant enrichment of differentially expressed genes within the Wnt signaling pathway. Further investigation revealed that deletion of YTHDF2 increased the half-life and expression of Dickkopf homolog 3 (DKK3), a potent inhibitor of the Wnt signaling pathway. Consequently, the inhibition of Wnt signaling attenuated the inflammatory response and inhibited cell proliferation.

DEAD-box RNA helicase 10 is required for 18S rRNA maturation by controlling the release of U3 snoRNA from pre-rRNA in embryonic stem cells

Ribosome biogenesis plays a pivotal role in maintaining stem cell homeostasis, yet the precise regulatory mechanisms governing this process in mouse embryonic stem cells (mESCs) remain largely unknown. In this investigation, we ascertain that DEAD-box RNA helicase 10 (DDX10) is indispensable for upholding cellular homeostasis and the viability of mESCs. Positioned predominantly at the nucleolar dense fibrillar component (DFC) and granular component (GC), DDX10 predominantly binds to 45S ribosomal RNA (rRNA) and orchestrates ribosome biogenesis. Degradation of DDX10 prevents the release of U3 snoRNA from pre-rRNA, leading to perturbed pre-rRNA processing and compromised maturation of the 18S rRNA, thereby disrupting the biogenesis of the small ribosomal subunit. Moreover, DDX10 participates in the process of liquid-liquid phase separation (LLPS), which is necessary for efficient ribosome biogenesis. Notably, the NUP98-DDX10 fusion associated with acute myelocytic leukemia (AML) alters the cellular localization of DDX10 and results in loss of ability to regulate pre-rRNA processing. Collectively, this study reveals the critical role of DDX10 as a pivotal regulator of ribosome biogenesis in mESCs.

LncRNAs maintain the functional phase state of nucleolar prion-like protein to facilitate rRNA processing.

Liquid-to-solid phase transition of proteins with prion-like domains (PLDs) has been associated with neurodegenerative diseases and aging. High protein concentration is one important aspect triggering the transition; however, several prion-like proteins, including fibrillarin (FBL), an important phase-separated protein in the nucleolus for pre-rRNA processing, show relatively high expression levels in certain cells, especially cancer cells, without obvious phase transitions and growth arrest. How cells maintain prion-like protein proteostasis is still unknown. Here, we attempt to answer the question, with FBL as an example. We find that lncRNA DNAJC3-AS1 can buffer the behavior of FBL condensation and maintain the state and function of fibrillar component/dense fibrillar component (FC/DFC) units in human cell lines through two mechanisms, not only facilitating FBL condensation but also inhibiting excessive aggregation by binding multiple PLDs and partially blocking their interactions. We propose that lncRNAs could supply buffered systems to sustain functional phase states of prion-like proteins.

Targeting BRIX1 via Engineered Exosomes Induces Nucleolar Stress to Suppress Cancer Progression.

Elevated ribosome biogenesis correlates with the rapid growth and progression of cancer. Targeted blockade of ribosome biogenesis induces nucleolar stress, which preferentially leads to the elimination of malignant cells. In this study, it is reported that the nucleolar protein BRIX1 is a critical regulator for the homeostasis between ribosome biogenesis and p53 activation. BRIX1 facilitated the processing of pre-rRNA by supporting the formation of the PeBoW complex. In addition, BRIX1 prevented p53 activation in response to nucleolar stress by impairing the interactions between MDM2 and the ribosomal proteins, RPL5, and RPL11, thereby triggering the resistance of cancer cells to chemotherapy. Conversely, depletion of BRIX1 induced nucleolar stress, which in turn activated p53 through RPL5 and RPL11, consequently inhibiting the growth of tumors. Moreover, engineered exosomes are developed, which are surface-decorated with iRGD, a tumor-homing peptide, and loaded with siRNAs specific to BRIX1, for the treatment of cancer. iRGD-Exo-siBRIX1 significantly suppressed the growth of colorectal cancer and enhanced the efficacy of 5-FU chemotherapy in vivo. Overall, the study uncovers that BRIX1 functions as an oncoprotein to promote rRNA synthesis and dampen p53 activity, and also implies that targeted inhibition of BRIX1 via engineered exosomes can be a potent approach for cancer therapy.

USP36 SUMOylates Las1L and Promotes Its Function in Pre-Ribosomal RNA ITS2 Processing.

This study identifies USP36 as a deubiquitinating and small ubiquitin-like modifier ligase dual-function enzyme to mediate Las1L deubiquitination and SUMOylation. Las1L SUMOylation at K565 plays a critical role in pre-rRNA ITS2 processing. Thus, our study reveals a novel downstream pathway for USP36-regulated ribosome biogenesis.

Peroxisomal import stress activates integrated stress response and inhibits ribosome biogenesis.

Impaired organelle-specific protein import triggers a variety of cellular stress responses, including adaptive pathways to balance protein homeostasis. Most of the previous studies focus on the cellular stress response triggered by misfolded proteins or defective protein import in the endoplasmic reticulum or mitochondria. However, little is known about the cellular stress response to impaired protein import in the peroxisome, an understudied organelle that has recently emerged as a key signaling hub for cellular and metabolic homeostasis. To uncover evolutionarily conserved cellular responses upon defective peroxisomal import, we carried out a comparative transcriptomic analysis on fruit flies with tissue-specific peroxin knockdown and human HEK293 cells expressing dominant-negative PEX5C11A. Our RNA-seq results reveal that defective peroxisomal import upregulates integrated stress response (ISR) and downregulates ribosome biogenesis in both flies and human cells. Functional analyses confirm that impaired peroxisomal import induces eIF2α phosphorylation and ATF4 expression. Loss of ATF4 exaggerates cellular damage upon peroxisomal import defects, suggesting that ATF4 activation serves as a cellular cytoprotective mechanism upon peroxisomal import stress. Intriguingly, we show that peroxisomal import stress decreases the expression of rRNA processing genes and inhibits early pre-rRNA processing, which leads to the accumulation of 47S precursor rRNA and reduction of downstream rRNA intermediates. Taken together, we identify ISR activation and ribosome biogenesis inhibition as conserved adaptive stress responses to defective peroxisomal import and uncover a novel link between peroxisomal dysfunction and rRNA processing.

Clinical and genetic delineation of autosomal recessive and dominant ACTL6B-related developmental brain disorders

PurposeThis study aims to comprehensively delineate the phenotypic spectrum of ACTL6B-related disorders, previously associated with both autosomal recessive and autosomal dominant neurodevelopmental disorders. Molecularly, the role of the nucleolar protein ACTL6B in contributing to the disease has remained unclear. MethodsWe identified 105 affected individuals, including 39 previously reported cases, and systematically analyzed detailed clinical and genetic data for all individuals. Additionally, we conducted knockdown experiments in neuronal cells to investigate the role of ACTL6B in ribosome biogenesis. ResultsBiallelic variants in ACTL6B are associated with severe-to-profound global developmental delay/intellectual disability, infantile intractable seizures, absent speech, autistic features, dystonia, and increased lethality. De novo monoallelic variants result in moderate-to-severe global developmental delay/intellectual disability, absent speech, and autistic features, whereas seizures and dystonia were less frequently observed. Dysmorphic facial features and brain abnormalities, including hypoplastic corpus callosum, and parenchymal volume loss/atrophy, are common findings in both groups. We reveal that in the nucleolus, ACTL6B plays a crucial role in ribosome biogenesis, particularly in pre-rRNA processing. ConclusionThis study provides a comprehensive characterization of the clinical spectrum of both autosomal recessive and dominant forms of ACTL6B-associated disorders. It offers a comparative analysis of their respective phenotypes provides a plausible molecular explanation and suggests their inclusion within the expanding category of “ribosomopathies.”

The novel ribosome biogenesis inhibitor usnic acid blocks nucleolar pre-60S maturation

The formation of new ribosomes is tightly coordinated with cell growth and proliferation. In eukaryotes, the correct assembly of all ribosomal proteins and RNAs follows an intricate scheme of maturation and rearrangement steps across three cellular compartments: the nucleolus, nucleoplasm, and cytoplasm. We demonstrate that usnic acid, a lichen secondary metabolite, inhibits the maturation of the large ribosomal subunit in yeast. We combine biochemical characterization of pre-ribosomal particles with a quantitative single-particle cryo-EM approach to monitor changes in nucleolar particle populations upon drug treatment. Usnic acid rapidly blocks the transition from nucleolar state B to C of Nsa1-associated pre-ribosomes, depleting key maturation factors such as Dbp10 and hindering pre-rRNA processing. This primary nucleolar block rapidly rebounds on earlier stages of the pathway which highlights the regulatory linkages between different steps. In summary, we provide an in-depth characterization of the effect of usnic acid on ribosome biogenesis, which may have implications for its reported anti-cancer activities.

Structural characterization of DNA-binding domain of essential mammalian protein TTF 1.

Transcription Termination Factor 1 (TTF1) is a multifunctional mammalian protein with vital roles in various cellular processes, including Pol I-mediated transcription initiation and termination, pre-rRNA processing, chromatin remodelling, DNA damage repair, and polar replication fork arrest. It comprises two distinct functional regions; the N-terminal regulatory region (1-445 aa), and the C-terminal catalytic region (445-859 aa). The Myb domain located at the C-terminal region is a conserved DNA binding domain spanning from 550 to 732 aa (183 residues). Despite its critical role in various cellular processes, the physical structure of TTF1 remains unsolved. Attempts to purify the functional TTF1 protein have been unsuccessful till date. Therefore, we focused on characterizing the Myb domain of this essential protein. We started with predicting a 3-D model of the Myb domain using homology modelling, and ab-initio method. We then determined its stability through MD simulation in an explicit solvent. The model predicted is highly stable, which stabilizes at 200ns. To experimentally validate the computational model, we cloned and expressed the codon optimized Myb domain into a bacterial expression vector and purified the protein to homogeneity. Further, characterization of the protein shows that, Myb domain is predominantly helical (65%) and is alone sufficient to bind the Sal Box DNA. This is the first-ever study to report a complete in silico model of the Myb domain, which is physically characterized. The above study will pave the way towards solving the atomic structure of this essential mammalian protein.

Maize Dek51 encodes a DEAD-box RNA helicase essential for pre-rRNA processing and seed development

Pre-rRNA processing is essential to ribosome biosynthesis. However, the processing mechanism is not fully understood in plants. Here, we report a DEAD-box RNA helicase DEK51 that mediates the 3' end processing of 18S and 5.8S pre-rRNA in maize (Zea mays L.). DEK51 is localized in the nucleolus, and loss of DEK51 arrests maize seed development and blocks the 3' end processing of 18S and 5.8S pre-rRNA. DEK51 interacts with putative key factors in nuclear RNA exosome-mediated pre-rRNA processing, including ZmMTR4, ZmSMO4, ZmRRP44A, and ZmRRP6L2. This suggests that DEK51 facilitates pre-rRNA processing by interacting with the exosome. Loss of ZmMTR4 function arrests seed development and blocks the 3' end processing of 18S and 5.8S pre-rRNA, similar to dek51. DEK51 also interacts with endonucleases ZmUTP24 and ZmRCL1, suggesting that it may also be involved in the cleavage at site A2. These results show the critical role of DEK51 in promoting 3' end processing of pre-rRNA.

Putting It All Together: The Roles of Ribosomal Proteins in Nucleolar Stages of 60S Ribosomal Assembly in the Yeast Saccharomyces cerevisiae.

Here we review the functions of ribosomal proteins (RPs) in the nucleolar stages of large ribosomal subunit assembly in the yeast Saccharomyces cerevisiae. We summarize the effects of depleting RPs on pre-rRNA processing and turnover, on the assembly of other RPs, and on the entry and exit of assembly factors (AFs). These results are interpreted in light of recent near-atomic-resolution cryo-EM structures of multiple assembly intermediates. Results are discussed with respect to each neighborhood of RPs and rRNA. We identify several key mechanisms related to RP behavior. Neighborhoods of RPs can assemble in one or more than one step. Entry of RPs can be triggered by molecular switches, in which an AF is replaced by an RP binding to the same site. To drive assembly forward, rRNA structure can be stabilized by RPs, including clamping rRNA structures or forming bridges between rRNA domains.

Reduced dynamicity and increased high-order protein assemblies in dense fibrillar component of the nucleolus under cellular senescence

Cellular senescence, which is triggered by various stressors, manifests as irreversible cell cycle arrest, resulting in the disruption of multiple nuclear condensates. One of the affected structures is the nucleolus, whose tripartite layout, separated into distinct liquid phases, allows for the stepwise progression of ribosome biogenesis. The dynamic properties of dense fibrillar components, a sub-nucleolar phase, are crucial for mediating pre-rRNA processing. However, the mechanistic link between the material properties of dense fibrillar components and cellular senescence remains unclear. We established a significant association between cellular senescence and alterations in nucleolar materiality and characteristics, including the number, size, and sphericity of individual subphases of the nucleolus. Senescent cells exhibit reduced fibrillarin dynamics, aberrant accumulation of high-order protein assemblies, such as oligomers and fibrils, and increased dense fibrillar component density. Intriguingly, the addition of RNA-interacting entities mirrored the diminished diffusion of fibrillarin in the nucleolus during cellular senescence. Thus, our findings contribute to a broader understanding of the intricate changes in the materiality of the nucleolus associated with cellular senescence and shed light on nucleolar dynamics in the context of aging and cellular stress.

Dotting Out AML by Targeting Fibrillarin.

Dysregulated biomolecular condensates, formed through multivalent interactions among proteins and nucleic acids, have been recently identified to drive tumorigenesis. In acute myeloid leukemia (AML), condensates driven by RNA-binding proteins alter transcriptional networks. Yang and colleagues performed a CRISPR screen and identified fibrillarin (FBL) as a new driver in AML leukemogenesis. FBL depletion caused cell cycle arrest and death in AML cells, with minimal impact on normal cells. FBL's phase separation domains are essential for pre-rRNA processing, influencing AML cell survival by regulating ribosome biogenesis and the translation of oncogenic proteins like MYC. Therapeutically, the chemotherapeutic agent CGX-635 targets FBL, inducing its aggregation, impairing pre-rRNA processing, and reducing AML cell survival. This highlights FBL's phase separation as a therapeutic vulnerability in AML. These findings suggest that targeting the phase separation properties of RNA-binding proteins could offer a novel and effective strategy for AML treatment. Further research into condensate dynamics in cancer and development of condensate-modulating drugs holds significant promise for future cancer therapies.

IGHMBP2 deletion suppresses translation and activates the integrated stress response.

IGHMBP2 is a nonessential, superfamily 1 DNA/RNA helicase that is mutated in patients with rare neuromuscular diseases SMARD1 and CMT2S. IGHMBP2 is implicated in translational and transcriptional regulation via biochemical association with ribosomal proteins, pre-rRNA processing factors, and tRNA-related species. To uncover the cellular consequences of perturbing IGHMBP2, we generated full and partial IGHMBP2 deletion K562 cell lines. Using polysome profiling and a nascent protein synthesis assay, we found that IGHMBP2 deletion modestly reduces global translation. We performed Ribo-seq and RNA-seq and identified diverse gene expression changes due to IGHMBP2 deletion, including ATF4 up-regulation. With recent studies showing the integrated stress response (ISR) can contribute to tRNA metabolism-linked neuropathies, we asked whether perturbing IGHMBP2 promotes ISR activation. We generated ATF4 reporter cell lines and found IGHMBP2 knockout cells demonstrate basal, chronic ISR activation. Our work expands upon the impact of IGHMBP2 in translation and elucidates molecular mechanisms that may link mutant IGHMBP2 to severe clinical phenotypes.

Phase separation-competent FBL promotes early pre-rRNA processing and translation in acute myeloid leukaemia.

RNA-binding proteins (RBPs) are pivotal in acute myeloid leukaemia (AML), a lethal disease. Although specific phase separation-competent RBPs are recognized in AML, the effect of their condensate formation on AML leukaemogenesis, and the therapeutic potential of inhibition of phase separation are underexplored. In our in vivo CRISPR RBP screen, fibrillarin (FBL) emerges as a crucial nucleolar protein that regulates AML cell survival, primarily through its phase separation domains rather than methyltransferase or acetylation domains. These phase separation domains, with specific features, coordinately drive nucleoli formation and early processing of pre-rRNA (including efflux, cleavage and methylation), eventually enhancing the translation of oncogenes such as MYC. Targeting the phase separation capability of FBL with CGX-635 leads to elimination of AML cells, suggesting an additional mechanism of action for CGX-635 that complements its established therapeutic effects. We highlight the potential of PS modulation of critical proteins as a possible therapeutic strategy for AML.

Yeast Crf1p is an activator with different roles in regulation of target genes.

Under stress conditions, ribosome biogenesis is downregulated. This process requires that expression of ribosomal RNA, ribosomal protein, and ribosome biogenesis genes be controlled in a coordinated fashion. The mechanistic Target of Rapamycin Complex 1 (mTORC1) participates in sensing unfavorable conditions to effect the requisite change in gene expression. In Saccharomyces cerevisiae, downregulation of ribosomal protein genes involves dissociation of the activator Ifh1p in a process that depends on Utp22p, a protein that also functions in pre-rRNA processing. Ifh1p has a paralog, Crf1p, which was implicated in communicating mTORC1 inhibition and hence was perceived as a repressor. We focus here on two ribosomal biogenesis genes, encoding Utp22p and the high mobility group protein Hmo1p, both of which are required for communication of mTORC1 inhibition to target genes. Crf1p functions as an activator on these genes as evidenced by reduced mRNA abundance and RNA polymerase II occupancy in a crf1Δ strain. Inhibition of mTORC1 has distinct effects on expression of HMO1 and UTP22; for example, on UTP22, but not on HMO1, the presence of Crf1p promotes the stable depletion of Ifh1p. Our data suggest that Crf1p functions as a weak activator, and that it may be required to prevent re-binding of Ifh1p to some gene promoters after mTORC1 inhibition in situations when Ifh1p is available. We propose that the inclusion of genes encoding proteins required for mTORC1-mediated downregulation of ribosomal protein genes in the same regulatory circuit as the ribosomal protein genes serves to optimize transcriptional responses during mTORC1 inhibition.

The novel pre-rRNA detection workflow "Riboprobing" allows simple identification of undescribed RNA species.

Ribosomes translate mRNA into proteins and are essential for every living organism. In eukaryotes, both ribosomal subunits are rapidly assembled in a strict hierarchical order, starting in the nucleolus with the transcription of a common precursor ribosomal RNA (pre-rRNA). This pre-rRNA encodes three of the four mature rRNAs, which are formed by several, consecutive endonucleolytic and exonucleolytic processing steps. Historically, northern blots are used to analyze the variety of different pre-rRNA species, only allowing rough length estimations. Although this limitation can be overcome with primer extension, both approaches often use radioactivity and are time-consuming and costly. Here, we present "Riboprobing," a linker ligation-based workflow followed by reverse transcription and PCR for easy and fast detection and characterization of pre-rRNA species and their 5' as well as 3' ends. Using standard molecular biology laboratory equipment, "Riboprobing" allows reliable discrimination of pre-rRNA species not resolved by northern blot (e.g., 27SA2, 27SA3, and 27SB pre-rRNA). The method can successfully be used for the analysis of total cell extracts as well as purified pre-ribosomes for a straightforward evaluation of the impact of mutant gene versions or inhibitors. In the course of method development, we identified and characterized a hitherto undescribed aberrant pre-rRNA arising from LiCl inhibition. This pre-rRNA fragment spans from processing site A1 to E, forming a small RNP that lacks most early joining assembly factors. This finding expands our knowledge of how the cell deals with severe pre-rRNA processing defects and demonstrates the strict requirement for the 5'ETS (external transcribed spacer) for the assembly process.

Dnttip2 is essential for 18S rRNA processing and digestive organ development in zebrafish

Dnttip2 is one of the components of the small subunit (SSU) processome. In yeast, depletion of dnttip2 leads to an inefficient processing of pre-rRNA and a decrease in synthesis of the mature 18S rRNA. However, the biological roles of Dnttip2 in higher organisms are poorly defined. In this study, we demonstrate that dnttip2 is a maternal gene in zebrafish. Depletion of Dnttip2 leads to embryonic lethal with severe digestive organs hypoplasia. The loss of function of Dnttip2 also leads to partial defects in cleavage at the A0-site and E-site during 18S rRNA processing. In conclusion, Dnttip2 is essential for 18S rRNA processing and digestive organ development in zebrafish.

Human RPF1 and ESF1 in Pre-rRNA Processing and the Assembly of Pre-Ribosomal Particles: A Functional Study.

Ribosome biogenesis is essential for the functioning of living cells. In higher eukaryotes, this multistep process is tightly controlled and involves a variety of specialized proteins and RNAs. This pool of so-called ribosome biogenesis factors includes diverse proteins with enzymatic and structural functions. Some of them have homologs in yeast S. cerevisiae, and their function can be inferred from the structural and biochemical data obtained for the yeast counterparts. The functions of human proteins RPF1 and ESF1 remain largely unclear, although RPF1 has been recently shown to participate in 60S biogenesis. Both proteins have drawn our attention since they contribute to the early stages of ribosome biogenesis, which are far less studied than the later stages. In this study, we employed the loss-of-function shRNA/siRNA-based approach to the human cell line HEK293 to determine the role of RPF1 and ESF1 in ribosome biogenesis. Downregulating RPF1 and ESF1 significantly changed the pattern of RNA products derived from 47S pre-rRNA. Our findings demonstrate that RPF1 and ESF1 are associated with different pre-ribosomal particles, pre-60S, and pre-40S particles, respectively. Our results allow for speculation about the particular steps of pre-rRNA processing, which highly rely on the RPF1 and ESF1 functions. We suggest that both factors are not directly involved in pre-rRNA cleavage but rather help pre-rRNA to acquire the conformation favoring its cleavage.

Monoallelically expressed noncoding RNAs form nucleolar territories on NOR-containing chromosomes and regulate rRNA expression.

Out of the several hundred copies of rRNA genes arranged in the nucleolar organizing regions (NOR) of the five human acrocentric chromosomes, ~50% remain transcriptionally inactive. NOR-associated sequences and epigenetic modifications contribute to the differential expression of rRNAs. However, the mechanism(s) controlling the dosage of active versus inactive rRNA genes within each NOR in mammals is yet to be determined. We have discovered a family of ncRNAs, SNULs (Single NUcleolus Localized RNA), which form constrained sub-nucleolar territories on individual NORs and influence rRNA expression. Individual members of the SNULs monoallelically associate with specific NOR-containing chromosomes. SNULs share sequence similarity to pre-rRNA and localize in the sub-nucleolar compartment with pre-rRNA. Finally, SNULs control rRNA expression by influencing pre-rRNA sorting to the DFC compartment and pre-rRNA processing. Our study discovered a novel class of ncRNAs influencing rRNA expression by forming constrained nucleolar territories on individual NORs.