Ribosomal RNA Processing Research Articles

The Inherited MDS Gene DDX41 Is Essential for Ribosomal RNA Processing through Regulation of Snorna Biogenesis

Germline mutations in the DEAD-box RNA helicase gene DDX41 cause inherited susceptibility to Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML). These patients have normal hematopoietic indices into adulthood and present with MDS at a median age of 61 years, slightly younger than the general MDS patient population (71 years). Germline DDX41 mutations are always heterozygous and are typically frame-shift mutations, causing loss of function of the protein. More than half of these patients acquire a second-hit mutation in the healthy DDX41 allele in their disease clones. Greater than 80% of the second-hit mutations cause the amino acid substitution R525H, which results in loss of helicase activity. Multiple functions have been ascribed to DDX41, such as functioning as an innate immune sensor, a component of the RNA spliceosome, and a regulator of ribosomal RNA processing; however, its role in the pathogenesis of MDS remains poorly understood. To mimic the DDX41 mutations in human patients, we generated conditional mouse models of the most common DDX41 mutations, D140fs and R525H. We found that DDX41 is essential for the viability and function of hematopoietic stem and progenitor cells (HSPCs) and that R525H mutations render DDX41 inactive. We have also reported that mice with biallelic DDX41 mutations develop progressive anemia, dysplasia, and marrow failure. HSPCs lacking wild-type Ddx41 have dysfunctional ribosomes and reduced protein translation, leading to cycle arrest and apoptosis. As a mechanistic basis for the ribosome defect in Ddx41-deficient HSPCs, we found that loss of Ddx41 causes a profound increase in unprocessed snoRNA transcripts, which likely interrupts their cellular function. Importantly, we did not observe a change in host gene transcript abundance, and thus it is unlikely that the defects observed in Ddx41-deficient cells are caused by loss of the protein products of the host genes. SnoRNAs regulate ribosomal RNA (rRNA) by catalyzing post-transcriptional modifications, including methylation and pseudouridylation. Since the majority of the snoRNAs that were found to be unprocessed in Ddx41-deficient HSPCs were from the SNORA family, which is involved in pseudouridylation, we quantified the abundance of pseudouridine at specific sites in ribosomal RNA and found it to be reduced in Ddx41-deficient HSPCs. Utilizing global high-throughput sequencing approaches to analyze DDX41 binding to RNAs, we found that DDX41 preferentially binds to snoRNAs relative to all other types of RNA in the cells. Thus, DDX41 binds to snoRNAs and is essential for snoRNA processing, snoRNA-mediated rRNA pseudouridylation, and protein translation in HSPCs. These findings provide critical mechanistic insight into the protein translation defect that we and others have observed in DDX41-deficient cells and uncover the basis of ineffective hematopoiesis in MDS patients with DDX41 mutations. Disclosures Starczynowski: Tolero Therapeutics: Research Funding; Kurome Therapeutics: Consultancy, Current equity holder in private company, Research Funding; Captor Therapeutics: Consultancy.

Ribosome biogenesis is a downstream effector of the oncogenic U2AF1-S34F mutation.

U2 Small Nuclear RNA Auxiliary Factor 1 (U2AF1) forms a heterodimeric complex with U2AF2 that is primarily responsible for 3ʹ splice site selection. U2AF1 mutations have been identified in most cancers but are prevalent in Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML), and the most common mutation is a missense substitution of serine-34 to phenylalanine (S34F). The U2AF heterodimer also has a noncanonical function as a translational regulator. Here, we report that the U2AF1-S34F mutation results in specific misregulation of the translation initiation and ribosome biogenesis machinery. The net result is an increase in mRNA translation at the single-cell level. Among the translationally up-regulated targets of U2AF1-S34F is Nucleophosmin 1 (NPM1), which is a major driver of myeloid malignancy. Depletion of NPM1 impairs the viability of the U2AF1-S34F mutant cells and causes ribosomal RNA (rRNA) processing defects, thus indicating an unanticipated synthetic interaction between U2AF1, NPM1, and ribosome biogenesis. Our results establish a unique molecular phenotype for the U2AF1 mutation that recapitulates translational misregulation in myeloid disease.

Ribosome Biogenesis Alterations in Colorectal Cancer.

Many studies have focused on understanding the regulation and functions of aberrant protein synthesis in colorectal cancer (CRC), leaving the ribosome, its main effector, relatively underappreciated in CRC. The production of functional ribosomes is initiated in the nucleolus, requires coordinated ribosomal RNA (rRNA) processing and ribosomal protein (RP) assembly, and is frequently hyperactivated to support the needs in protein synthesis essential to withstand unremitting cancer cell growth. This elevated ribosome production in cancer cells includes a strong alteration of ribosome biogenesis homeostasis that represents one of the hallmarks of cancer cells. None of the ribosome production steps escape this cancer-specific dysregulation. This review summarizes the early and late steps of ribosome biogenesis dysregulations described in CRC cell lines, intestinal organoids, CRC stem cells and mouse models, and their possible clinical implications. We highlight how this cancer-related ribosome biogenesis, both at quantitative and qualitative levels, can lead to the synthesis of ribosomes favoring the translation of mRNAs encoding hyperproliferative and survival factors. We also discuss whether cancer-related ribosome biogenesis is a mere consequence of cancer progression or is a causal factor in CRC, and how altered ribosome biogenesis pathways can represent effective targets to kill CRC cells. The association between exacerbated CRC cell growth and alteration of specific steps of ribosome biogenesis is highlighted as a key driver of tumorigenesis, providing promising perspectives for the implementation of predictive biomarkers and the development of new therapeutic drugs.

WUSCHEL triggers innate antiviral immunity in plant stem cells.

Stem cells in plants constantly supply daughter cells to form new organs and are expected to safeguard the integrity of the cells from biological invasion. Here, we show how stem cells of the Arabidopsis shoot apical meristem and their nascent daughter cells suppress infection by cucumber mosaic virus (CMV). The stem cell regulator WUSCHEL responds to CMV infection and represses virus accumulation in the meristem central and peripheral zones. WUSCHEL inhibits viral protein synthesis by repressing the expression of plant S-adenosyl-l-methionine-dependent methyltransferases, which are involved in ribosomal RNA processing and ribosome stability. Our results reveal a conserved strategy in plants to protect stem cells against viral intrusion and provide a molecular basis for WUSCHEL-mediated broad-spectrum innate antiviral immunity in plants.

Building a safe space for stem cells

Plant Science The meristem, the collection of stem cells that builds plants, is resistant to viral infection. Wu et al. now show that WUSCHEL, a transcription factor that helps to sustain stem cell production in the shoot apical meristem of Arabidopsis , also protects that stem cell domain from viruses. WUSCHEL inhibited viral protein synthesis by repressing methyltransferases that regulate ribosomal RNA processing and ribosome stability. Science , this issue p. [227][1] [1]: /lookup/doi/10.1126/science.abb7360

A Type IA DNA/RNA Topoisomerase with RNA Hydrolysis Activity Participates in Ribosomal RNA Processing

Topoisomerases maintain topological homeostasis of bacterial chromosomes by catalysing changes in DNA linking number. The resolution of RNA entanglements occurring in the cell would also require catalytic action of topoisomerases. We describe RNA topoisomerase and hydrolysis activities in DNA topoisomerase I (topo I) from mycobacteria. The interaction of topo I with mRNA, tRNA and rRNA suggested its role in some aspect of RNA metabolism; the enzyme participates in rRNA maturation via its RNA hydrolysis activity. Accumulation of rRNA precursors in a topo I knockdown strain and the rescue of rRNA processing deficiency in RNaseE knockdown cells by topo I expression indicated the enzyme’s back-up support to RNases involved in rRNA processing. We demonstrate that the active-site tyrosine of the enzyme mediates catalytic reactions with both DNA/RNA substrates, and RNA topoisomerase activity can follow two reaction paths in contrast to its DNA topoisomerase activity. Mutation in the canonical proton relay pathway impacts DNA topoisomerase activity whilst retaining activity on RNA substrates. The mycobacterial topo I thus exemplifies the resourcefulness and parsimony of biological catalysis in harnessing the limited chemical repertoire at its disposal to find common solutions to mechanistically related challenges of phosphodiester breakage/exchange reactions in DNA and RNA that are essential for cell survival.

Cryo-EM reveals active site coordination within a multienzyme pre-rRNA processing complex

Ribosome assembly is a complex process reliant on the coordination of trans-acting enzymes to produce functional ribosomal subunits and secure the translational capacity of cells. The endoribonuclease (RNase) Las1 and the polynucleotide kinase (PNK) Grc3 assemble into a multienzyme complex, herein designated RNase PNK, to orchestrate processing of precursor ribosomal RNA (rRNA). RNase PNK belongs to the functionally diverse HEPN nuclease superfamily, whose members rely on distinct cues for nuclease activation. To establish how RNase PNK coordinates its dual enzymatic activities, we solved a series of cryo-EM structures of Chaetomium thermophilum RNase PNK in multiple conformational states. The structures reveal that RNase PNK adopts a butterfly-like architecture, harboring a composite HEPN nuclease active site flanked by discrete RNA kinase sites. We identify two molecular switches that coordinate nuclease and kinase function. Together, our structures and corresponding functional studies establish a new mechanism of HEPN nuclease activation essential for ribosome production.

3060 – DOWNREGULATED STAUFEN1 COMPROMISES HEMATOPOIETIC STEM CELL ACTIVITY IN VIVO AND ELICITS EXPRESSION SIGNATURES CHARACTERISTIC OF CLINICAL ANEMIAS

Proper regulation of RNA localization, fidelity and translation are fundamental mechanisms regulated by RNA-binding proteins (RBPs). Staufen, a double-stranded RBP, mediates asymmetric cell division in developing neural precursors; however, its role in normal hematopoiesis remains unclear. Here, we studied the role of both mammalian paralogs of Staufen and identify that Stau1, but not Stau2, is enriched in both hematopoietic stem cell (HSC) and erythroid populations and is functionally essential to HSC activity in vivo post-transplantation. Unbiased global profiling of transcriptome and proteome changes with Stau1 downregulation in the primitive hematopoietic compartment reveal expression signatures predictive of an immune/inflammatory-associated anemia, with post-transcriptional pathway analyses highlighting concordant loss of erythroid-promoting transcription factors and cell cycle regulators, gain of an expression state consistent with interferon-γ activity, enhanced levels of translational regulation and decreased ribosomal RNA processing. These findings position Stau1 as a potential guardian of HSC-driven erythropoiesis. Our data further highlight a rational foundation for future investigations into the role of Stau1 in aplastic anemia and/or paroxysmal nocturnal hemoglobinuria, and may widen the scope of post-transcriptional gene regulation into anemia pathogenesis.

The Diabetes Gene JAZF1 Is Essential for the Homeostatic Control of Ribosome Biogenesis and Function in Metabolic Stress

The ability of pancreatic β-cells to respond to increased demands for insulin during metabolic stress critically depends on proper ribosome homeostasis and function. Excessive and long-lasting stimulation of insulin secretion can elicit endoplasmic reticulum (ER) stress, unfolded protein response, and β-cell apoptosis. Here we show that the diabetes susceptibility gene JAZF1 is a key transcriptional regulator of ribosome biogenesis, global protein, and insulin translation. JAZF1 is excluded from the nucleus, and its expression levels are reduced upon metabolic stress and in diabetes. Genetic deletion of Jazf1 results in global impairment of protein synthesis that is mediated by defects in ribosomal protein synthesis, ribosomal RNA processing, and aminoacyl-synthetase expression, thereby inducing ER stress and increasing β-cell susceptibility to apoptosis. Importantly, JAZF1 function and its pleiotropic actions are impaired in islets of murine T2D and in human islets exposed to metabolic stress. Our study identifies JAZF1 as a central mediator of metabolic stress in β-cells.

Structural insight into precursor ribosomal RNA processing by ribonuclease MRP.

Ribonuclease (RNase) MRP is a conserved eukaryotic ribonucleoprotein complex that plays essential roles in precursor ribosomal RNA (pre-rRNA) processing and cell cycle regulation. In contrast to RNase P, which selectively cleaves transfer RNA-like substrates, it has remained a mystery how RNase MRP recognizes its diverse substrates. To address this question, we determined cryo-electron microscopy structures of Saccharomyces cerevisiae RNase MRP alone and in complex with a fragment of pre-rRNA. These structures and the results of biochemical studies reveal that coevolution of both protein and RNA subunits has transformed RNase MRP into a distinct ribonuclease that processes single-stranded RNAs by recognizing a short, loosely defined consensus sequence. This broad substrate specificity suggests that RNase MRP may have myriad yet unrecognized substrates that could play important roles in various cellular contexts.

Prefoldin-like Bud27 influences the transcription of ribosomal components and ribosome biogenesis in Saccharomyces cerevisiae.

Understanding the functional connection that occurs for the three nuclear RNA polymerases to synthesize ribosome components during the ribosome biogenesis process has been the focal point of extensive research. To preserve correct homeostasis on the production of ribosomal components, cells might require the existence of proteins that target a common subunit of these RNA polymerases to impact their respective activities. This work describes how the yeast prefoldin-like Bud27 protein, which physically interacts with the Rpb5 common subunit of the three RNA polymerases, is able to modulate the transcription mediated by the RNA polymerase I, likely by influencing transcription elongation, the transcription of the RNA polymerase III, and the processing of ribosomal RNA. Bud27 also regulates both RNA polymerase II-dependent transcription of ribosomal proteins and ribosome biogenesis regulon genes, likely by occupying their DNA ORFs, and the processing of the corresponding mRNAs. With RNA polymerase II, this association occurs in a transcription rate-dependent manner. Our data also indicate that Bud27 inactivation alters the phosphorylation kinetics of ribosomal protein S6, a readout of TORC1 activity. We conclude that Bud27 impacts the homeostasis of the ribosome biogenesis process by regulating the activity of the three RNA polymerases and, in this way, the synthesis of ribosomal components. This quite likely occurs through a functional connection of Bud27 with the TOR signaling pathway.

A conserved RNA degradation complex required for spreading and epigenetic inheritance of heterochromatin.

Heterochromatic domains containing histone H3 lysine 9 methylation (H3K9me) can be epigenetically inherited independently of underlying DNA sequence. To gain insight into the mechanisms that mediate epigenetic inheritance, we used a Schizosaccharomyces pombe inducible heterochromatin formation system to perform a genetic screen for mutations that abolish heterochromatin inheritance without affecting its establishment. We identified mutations in several pathways, including the conserved and essential Rix1-associated complex (henceforth the rixosome), which contains RNA endonuclease and polynucleotide kinase activities with known roles in ribosomal RNA processing. We show that the rixosome is required for spreading and epigenetic inheritance of heterochromatin in fission yeast. Viable rixosome mutations that disrupt its association with Swi6/HP1 fail to localize to heterochromatin, lead to accumulation of heterochromatic RNAs, and block spreading of H3K9me and silencing into actively transcribed regions. These findings reveal a new pathway for degradation of heterochromatic RNAs with essential roles in heterochromatin spreading and inheritance.

Pseudouridine Synthase RsuA Captures an Assembly Intermediate that Is Stabilized by Ribosomal Protein S17.

The ribosome is a large ribonucleoprotein complex that synthesizes protein in all living organisms. Ribosome biogenesis is a complex process that requires synchronization of various cellular events, including ribosomal RNA (rRNA) transcription, ribosome assembly, and processing and post-transcriptional modification of rRNA. Ribosome biogenesis is fine-tuned with various assembly factors, possibly including nucleotide modification enzymes. Ribosomal small subunit pseudouridine synthase A (RsuA) pseudouridylates U516 of 16S helix 18. Protein RsuA is a multi-domain protein that contains the N-terminal peripheral domain, which is structurally similar to the ribosomal protein S4. Our study shows RsuA preferably binds and pseudouridylates an assembly intermediate that is stabilized by ribosomal protein S17 over the native-like complex. In addition, the N-terminal domain truncated RsuA showed that the presence of the S4-like domain is important for RsuA substrate recognition.

The in vivo functions of ARPF2 and ARRS1 in ribosomal RNA processing and ribosome biogenesis in Arabidopsis.

Yeast Rpf2 plays a critical role in the incorporation of 5S rRNA into pre-ribosomes by forming a binary complex with Rrs1. The protein characteristics and overexpression phenotypes of Arabidopsis Ribosome Production Factor 2 (ARPF2) and Arabidopsis Regulator of Ribosome Synthesis 1 (ARRS1) have been previously studied. Here, we analyze loss-of-function phenotypes of ARPF2 and ARRS1 using virus-induced gene silencing to determine their functions in pre-rRNA processing and ribosome biogenesis. ARPF2 silencing in Arabidopsis led to pleiotropic developmental defects. RNA gel blot analysis and circular reverse transcription-PCR revealed that ARPF2 depletion delayed pre-rRNA processing, resulting in the accumulation of multiple processing intermediates. ARPF2 fractionated primarily with the 60S ribosomal subunit. Metabolic rRNA labeling and ribosome profiling suggested that ARPF2 deficiency mainly affected 25S rRNA synthesis and 60S ribosome biogenesis. ARPF2 and ARRS1 formed the complex that interacted with the 60S ribosomal proteins RPL5 and RPL11. ARRS1 silencing resulted in growth defects, accumulation of processing intermediates, and ribosome profiling similar to those of ARPF2-silenced plants. Moreover, depletion of ARPF2 and ARRS1 caused nucleolar stress. ARPF2-deficient plants excessively accumulated anthocyanin and reactive oxygen species. Collectively, these results suggest that the ARPF2-ARRS1 complex plays a crucial role in plant growth and development by modulating ribosome biogenesis.

UL3 Mediated Nucleolar Stress Pathway as a New Mechanism of Action of Antiproliferative G-quadruplex TBA Derivatives in Colon Cancer Cells.

The antiproliferative G-quadruplex aptamers are a promising and challenging subject in the framework of the anticancer therapeutic oligonucleotides research field. Although several antiproliferative G-quadruplex aptamers have been identified and proven to be effective on different cancer cell lines, their mechanism of action is still unexplored. We have recently described the antiproliferative activity of a heterochiral thrombin binding aptamer (TBA) derivative, namely, LQ1. Here, we investigate the molecular mechanisms of LQ1 activity and the structural and antiproliferative properties of two further TBA derivatives, differing from LQ1 only by the small loop base-compositions. We demonstrate that in p53 deleted colon cancer cells, LQ1 causes nucleolar stress, impairs ribosomal RNA processing, leading to the accumulation of pre-ribosomal RNAs, arrests cells in the G2/M phase and induces early apoptosis. Importantly, the depletion of uL3 abrogates all these effects, indicating that uL3 is a crucial player in the mechanism of action of LQ1. Taken together, our findings identify p53-independent and uL3-dependent nucleolar stress as a novel stress response pathway activated by a specific G-quadruplex TBA derivative. To the best of our knowledge, this investigation reveals, for the first time, the involvement of the nucleolar stress pathway in the mechanism of action of antiproliferative G-quadruplex aptamers.

Principles of RNA processing from analysis of enhanced CLIP maps for 150 RNA binding proteins

BackgroundA critical step in uncovering rules of RNA processing is to study the in vivo regulatory networks of RNA binding proteins (RBPs). Crosslinking and immunoprecipitation (CLIP) methods enable mapping RBP targets transcriptome-wide, but methodological differences present challenges to large-scale analysis across datasets. The development of enhanced CLIP (eCLIP) enabled the mapping of targets for 150 RBPs in K562 and HepG2, creating a unique resource of RBP interactomes profiled with a standardized methodology in the same cell types.ResultsOur analysis of 223 eCLIP datasets reveals a range of binding modalities, including highly resolved positioning around splicing signals and mRNA untranslated regions that associate with distinct RBP functions. Quantification of enrichment for repetitive and abundant multicopy elements reveals 70% of RBPs have enrichment for non-mRNA element classes, enables identification of novel ribosomal RNA processing factors and sites, and suggests that association with retrotransposable elements reflects multiple RBP mechanisms of action. Analysis of spliceosomal RBPs indicates that eCLIP resolves AQR association after intronic lariat formation, enabling identification of branch points with single-nucleotide resolution, and provides genome-wide validation for a branch point-based scanning model for 3′ splice site recognition. Finally, we show that eCLIP peak co-occurrences across RBPs enable the discovery of novel co-interacting RBPs.ConclusionsThis work reveals novel insights into RNA biology by integrated analysis of eCLIP profiling of 150 RBPs with distinct functions. Further, our quantification of both mRNA and other element association will enable further research to identify novel roles of RBPs in regulating RNA processing.

Identification of RNA Exosome Cofactors in Neuronal Cells to Probe Disease Mechanism

The RNA exosome, a 10‐subunit complex that mediates both RNA processing and degradation, plays a critical role in defining cellular expression profiles. This complex is ubiquitously expressed, essential, and critical for fundamental cellular functions, such as ribosomal RNA processing. Recent studies have linked mutations in genes encoding multiple subunits of the complex to tissue‐specific human disease. For example, missense mutations in the human EXOSC3 gene, which encodes an RNA exosome subunit, cause Pontocerebellar Hypoplasia type 1b (PCH1b), a disease characterized by atrophy of the pons and cerebellum. The missense mutations encode single amino acid changes in conserved regions of the EXOSC3 protein. How these amino acid substitutions confer tissue‐specific phenotypes is not known. One possible mechanism underlying the distinct disease phenotypes could be a decrease in the interaction of the RNA exosome complex with cofactors that confer specificity for RNA targets. However, most studies that identify and characterize RNA exosome cofactors have been carried out in budding yeast and thus, do not provide insight into whether tissue‐specific cofactors could exist. Our studies use immunoprecipitation from neuronal cell culture (N2A) and relevant mouse tissues to define RNA exosome cofactors. Biochemical experiments that employ cultured N2A cells were used to identify RNA exosome‐interacting proteins in both the nucleus and the cytoplasm. Preliminary results from this analysis reveal an association between the RNA exosome and a large complex of cytoplasmic tRNA ligase enzymes, which could link defects in tRNA maturation to disease pathology. We are extending these studies to explore the possibility of tissue‐specific cofactors by immunoprecipitating EXOSC3 and analyzing co‐purifying proteins from the mouse cerebellum (affected in disease) and cortex (unaffected). To complement these approaches to identify tissue‐specific RNA exosome cofactors, we are also exploring whether amino acid changes that are linked to disease alter cofactor interactions. These studies, which are being performed in N2A cells, have identified cofactors that show altered interactions with the EXOSC3 variants present in disease. These studies will provide insight into both the functional consequences of amino acid substitutions in the RNA exosome that cause disease and the role of cofactors in conferring RNA target specificity.

Chemosensitization of prostate cancer stem cells in mice by angiogenin and plexin-B2 inhibitors

Cancer stem cells (CSCs) are an obstacle in cancer therapy and are a major cause of drug resistance, cancer recurrence, and metastasis. Available treatments, targeting proliferating cancer cells, are not effective in eliminating quiescent CSCs. Identification of CSC regulators will help design therapeutic strategies to sensitize drug-resistant CSCs for chemo-eradication. Here, we show that angiogenin and plexin-B2 regulate the stemness of prostate CSCs, and that inhibitors of angiogenin/plexin-B2 sensitize prostate CSCs to chemotherapy. Prostate CSCs capable of self-renewal, differentiation, and tumor initiation with a single cell inoculation were identified and shown to be regulated by angiogenin/plexin-B2 that promotes quiescence and self-renewal through 5S ribosomal RNA processing and generation of the bioactive 3′-end fragments of 5S ribosomal RNA, which suppress protein translation and restrict cell cycling. Monoclonal antibodies of angiogenin and plexin-B2 decrease the stemness of prostate CSCs and sensitize them to chemotherapeutic agents in vitro and in vivo.

Overexpressed L20 Rescues 50S Ribosomal Subunit Assembly Defects of bipA-Deletion in Escherichia coli.

The BipA (BPI-inducible protein A) protein is highly conserved in a large variety of bacteria and belongs to the translational GTPases, based on sequential and structural similarities. Despite its conservation in bacteria, bipA is not essential for cell growth under normal growth conditions. However, at 20°C, deletion of bipA causes not only severe growth defects but also several phenotypic changes such as capsule production, motility, and ribosome assembly, indicating that it has global regulatory properties. Our recent studies revealed that BipA is a novel ribosome-associating GTPase, whose expression is cold-shock-inducible and involved in the incorporation of the ribosomal protein (r-protein) L6. However, the precise mechanism of BipA in 50S ribosomal subunit assembly is not completely understood. In this study, to demonstrate the role of BipA in the 50S ribosomal subunit and possibly to find an interplaying partner(s), a genomic library was constructed and suppressor screening was conducted. Through screening, we found a suppressor gene, rplT, encoding r-protein L20, which is assembled at the early stage of ribosome assembly and negatively regulates its own expression at the translational level. We demonstrated that the exogenous expression of rplT restored the growth of bipA-deleted strain at low temperature by partially recovering the defects in ribosomal RNA processing and ribosome assembly. Our findings suggest that the function of BipA is pivotal for 50S ribosomal subunit biogenesis at a low temperature and imply that BipA and L20 may exert coordinated actions for proper ribosome assembly under cold-shock conditions.

STCH4/REIL2 Confers Cold Stress Tolerance in Arabidopsis by Promoting rRNA Processing and CBF Protein Translation

Plants respond to cold stress by inducing the expression of transcription factors that regulate downstream genes to confer tolerance to freezing. We screened an Arabidopsis transfer DNA (T-DNA) insertion library and identified a cold-hypersensitive mutant, which we named stch4 (sensitive to chilling 4). STCH4/REIL2 encodes a ribosomal biogenesis factor that is upregulated upon cold stress. Overexpression of STCH4 confers chilling and freezing tolerance in Arabidopsis. The stch4 mutation reduces CBF protein levels and thus delayed the induction of C-repeat-binding factor (CBF) regulon genes. Ribosomal RNAprocessing is reduced in stch4 mutants, especially under cold stress. STCH4 associates with multiple ribosomal proteins, and these interactions are modulated by cold stress. These results suggest that the ribosome is a regulatory node for cold stress responses and that STCH4 promotes an altered ribosomal composition and functions in low temperatures to facilitate the translation of proteins important for plant growth and survival under cold stress.