ZAKα Recognizes Stalled Ribosomes through Partially Redundant Sensor Domains.

Impairment of ribosome function activates the MAPKKK ZAK, leading to activation of mitogen-activated protein (MAP) kinases p38 and JNK and inflammatory signaling. The mechanistic basis for activation of this ribotoxic stress response (RSR) remains completely obscure. We show that the long isoform of ZAK (ZAKα) directly associates with ribosomes by inserting its flexible C terminus into the ribosomal intersubunit space. Here, ZAKα binds helix 14 of 18S ribosomal RNA (rRNA). An adjacent domain in ZAKα also probes the ribosome, and together, these sensor domains are critically required for RSR activation after inhibition of both the E-site, the peptidyl transferase center (PTC), and ribotoxin action. Finally, we show that ablation of the RSR response leads to organismal phenotypes and decreased lifespan in the nematode Caenorhabditis elegans (C. elegans). Our findings yield mechanistic insight into how cells detect ribotoxic stress and provide experimental in vivo evidence for its physiological importance.

GPATCH4 regulates rRNA and snRNA 2'-O-methylation in both DHX15-dependent and DHX15-independent manners.

Regulation of RNA helicase activity, often accomplished by protein cofactors, is essential to ensure target specificity within the complex cellular environment. The largest family of RNA helicase cofactors are the G-patch proteins, but the cognate RNA helicases and cellular functions of numerous human G-patch proteins remain elusive. Here, we discover that GPATCH4 is a stimulatory cofactor of DHX15 that interacts with the DEAH box helicase in the nucleolus via residues in its G-patch domain. We reveal that GPATCH4 associates with pre-ribosomal particles, and crosslinks to the transcribed ribosomal DNA locus and precursor ribosomal RNAs as well as binding to small nucleolar- and small Cajal body-associated RNAs that guide rRNA and snRNA modifications. Loss of GPATCH4 impairs 2'-O-methylation at various rRNA and snRNA sites leading to decreased protein synthesis and cell growth. We demonstrate that the regulation of 2'-O-methylation by GPATCH4 is both dependent on, and independent of, its interaction with DHX15. Intriguingly, the ATPase activity of DHX15 is necessary for efficient methylation of DHX15-dependent sites, suggesting a function of DHX15 in regulating snoRNA-guided 2'-O-methylation of rRNA that requires activation by GPATCH4. Overall, our findings extend knowledge on RNA helicase regulation by G-patch proteins and also provide important new insights into the mechanisms regulating installation of rRNA and snRNA modifications, which are essential for ribosome function and pre-mRNA splicing.

The rRNA epitranscriptome and myonuclear SNORD landscape in skeletal muscle fibers contributes to ribosome heterogeneity and is altered by hypertrophic stimulus.

In cell biology, ribosomal RNA (rRNA) 2'O-methyl (2'-O-Me) is the most prevalent post-transcriptional chemical modification contributing to ribosome heterogeneity. The modification involves a family of small nucleolar RNAs (snoRNAs) and is specified by box C/D snoRNAs (SNORDs). Given the importance of ribosome biogenesis for skeletal muscle growth, we asked if rRNA 2'-O-Me in nascent ribosomes synthesized in response to a growth stimulus is an unrecognized mode of ribosome heterogeneity in muscle. To determine the pattern and dynamics of 2'-O-Me rRNA, we used a sequencing-based profiling method called RiboMeth-seq. We applied this method to tissue-derived rRNA of skeletal muscle and rRNA specifically from the muscle fiber using an inducible myofiber-specific RiboTag mouse in sedentary and mechanically overloaded conditions. These analyses were complemented by myonuclear-specific small RNA sequencing to profile SNORDs and link the rRNA epitranscriptome to known regulatory elements generated within the muscle fiber. We demonstrate for the first time that mechanical overload of skeletal muscle 1) induces decreased 2'-O-Me at a subset of skeletal muscle rRNAand 2) alters the SNORD profile in isolated myonuclei. These findings point to a transient diversification of the ribosome pool via 2'-O-Me during growth and adaptation in skeletal muscle. These findings suggest changes in ribosome heterogeneity at the 2'-O-Me level during muscle hypertrophy and lay the foundation for studies investigating the functional implications of these newly identified "growth-induced" ribosomes.

RNA helicase-mediated regulation of snoRNP dynamics on pre-ribosomes and rRNA 2'-O-methylation.

RNA helicases play important roles in diverse aspects of RNA metabolism through their functions in remodelling ribonucleoprotein complexes (RNPs), such as pre-ribosomes. Here, we show that the DEAD box helicase Dbp3 is required for efficient processing of the U18 and U24 intron-encoded snoRNAs and 2'-O-methylation of various sites within the 25S ribosomal RNA (rRNA) sequence. Furthermore, numerous box C/D snoRNPs accumulate on pre-ribosomes in the absence of Dbp3. Many snoRNAs guiding Dbp3-dependent rRNA modifications have overlapping pre-rRNA basepairing sites and therefore form mutually exclusive interactions with pre-ribosomes. Analysis of the distribution of these snoRNAs between pre-ribosome-associated and 'free' pools demonstrated that many are almost exclusively associated with pre-ribosomal complexes. Our data suggest that retention of such snoRNPs on pre-ribosomes when Dbp3 is lacking may impede rRNA 2'-O-methylation by reducing the recycling efficiency of snoRNPs and by inhibiting snoRNP access to proximal target sites. The observation of substoichiometric rRNA modification at adjacent sites suggests that the snoRNPs guiding such modifications likely interact stochastically rather than hierarchically with their pre-rRNA target sites. Together, our data provide new insights into the dynamics of snoRNPs on pre-ribosomal complexes and the remodelling events occurring during the early stages of ribosome assembly.

The shift from early to late types of ribosomes in zebrafish development involves changes at a subset of rRNA 2'-O-Me sites.

During zebrafish development, an early type of rRNA is gradually replaced by a late type that is substantially different in sequence. We applied RiboMeth-seq to rRNA from developmental stages for profiling of 2'-O-Me, to learn if changes in methylation pattern were a component of the shift. We compiled a catalog of 2'-O-Me sites and cognate box C/D guide RNAs comprising 98 high-confidence sites, including 10 sites that were not known from other vertebrates, one of which was specific to late-type rRNA. We identified a subset of sites that changed in methylation status during development and found that some of these could be explained by availability of their cognate SNORDs. Sites that changed during development were enriched in the novel sites revealed in zebrafish. We propose that the early type of rRNA is a specialized form and that its structure and ribose methylation pattern may be an adaptation to features of development, including translation of specific maternal mRNAs.

Developmental changes of rRNA ribose methylations in the mouse.

A sequencing-based profiling method (RiboMeth-seq) for ribose methylations was used to study methylation patterns in mouse adult tissues and during development. In contrast to previous reports based on studies of human cancer cell lines, almost all methylation sites were close to fully methylated in adult tissues. A subset of sites was differentially modified in developing tissues compared to their adult counterparts and showed clear developmental dynamics. This provides the first evidence for ribosome heterogeneity at the level of rRNA modifications during mouse development. In a prominent example, the expression levels of SNORD78 during development appeared to be regulated by alternative splicing of the Gas5 host-gene and to correlate with the methylation level of its target site at LSU-G4593. The results are discussed in the context of the specialized ribosome hypothesis.

Sequencing-based methods for detection and quantitation of ribose methylations in RNA.

Ribose methylation is one of the most abundant RNA modifications and is found in all domains of life and all major classes of RNA (rRNA, tRNA, and mRNA). Ribose methylations are introduced by stand-alone enzymes or by generic enzymes guided to the target by small RNA guides. Recent years have seen the development of several sequencing-based methods for RNA modifications relying on different principles. In this review, we compare mapping and quantitation studies of ribose methylations from yeast and human culture cells. The emphasis is on ribosomal RNA for which the results can be compared to results from RNA fingerprinting and mass spectrometry. One sequencing approach is consistent with these methods and paints a conservative picture of rRNA modifications. Other approaches detect many more sites. Similar discrepancies are found in measurements of modification stoichiometry. The results are discussed in relation to the more challenging task of mapping ribose methylations in mRNA.

Lariat capping as a tool to manipulate the 5' end of individual yeast mRNA species in vivo.

The 5' cap structure of eukaryotic mRNA is critical for its processing, transport, translation, and stability. The many functions of the cap and the fact that most, if not all, mRNA carries the same type of cap makes it difficult to analyze cap function in vivo at individual steps of gene expression. We have used the lariat capping ribozyme (LCrz) from the myxomycete Didymium to replace the mRNA m7G cap of a single reporter mRNA species with a tiny lariat in which the first and the third nucleotide are joined by a 2', 5' phosphodiester bond. We show that the ribozyme functions in vivo in the budding yeast Saccharomyces cerevisiae presumably without cofactors and that lariat capping occurs cotranscriptionally. The lariat-capped reporter mRNA is efficiently exported to the cytoplasm where it is found to be oligoadenylated and evenly distributed. Both the oligoadenylated form and a lariat-capped mRNA with a templated poly(A) tail translates poorly, underlining the critical importance of the m7G cap in translation. Finally, the lariat-capped RNA exhibits a threefold longer half-life compared to its m7G-capped counterpart, consistent with a key role for the m7G cap in mRNA turnover. Our study emphasizes important activities of the m7G cap and suggests new utilities of lariat capping as a molecular tool in vivo.

Profiling of 2'-O-Me in human rRNA reveals a subset of fractionally modified positions and provides evidence for ribosome heterogeneity.

Ribose methylation is one of the two most abundant modifications in human ribosomal RNA and is believed to be important for ribosome biogenesis, mRNA selectivity and translational fidelity. We have applied RiboMeth-seq to rRNA from HeLa cells for ribosome-wide, quantitative mapping of 2'-O-Me sites and obtained a comprehensive set of 106 sites, including two novel sites, and with plausible box C/D guide RNAs assigned to all but three sites. We find approximately two-thirds of the sites to be fully methylated and the remainder to be fractionally modified in support of ribosome heterogeneity at the level of RNA modifications. A comparison to HCT116 cells reveals similar 2'-O-Me profiles with distinct differences at several sites. This study constitutes the first comprehensive mapping of 2'-O-Me sites in human rRNA using a high throughput sequencing approach. It establishes the existence of a core of constitutively methylated positions and a subset of variable, potentially regulatory positions, and paves the way for experimental analyses of the role of variations in rRNA methylation under different physiological or pathological settings.

Substoichiometric ribose methylations in spliceosomal snRNAs.

Sequencing-based profiling of ribose methylations is a new approach that allows for experiments addressing dynamic changes on a large scale. Here, we apply such a method to spliceosomal snRNAs present in human whole cell RNA. Analysis of solid tissue samples confirmed all previously known sites and demonstrated close to full methylation at almost all sites. Methylation changes were revealed in biological experimental settings, using T cell activation as an example, and in the T cell leukemia model, Jurkat cells. Such changes could impact the dynamics of snRNA interactions during the spliceosome cycle and affect mRNA splicing efficiency and splicing patterns.

Profiling of ribose methylations in RNA by high-throughput sequencing.

Ribose methylations are the most abundant chemical modifications of ribosomal RNA and are critical for ribosome assembly and fidelity of translation. Many aspects of ribose methylations have been difficult to study due to lack of efficient mapping methods. Here, we present a sequencing-based method (RiboMeth-seq) and its application to yeast ribosomes, presently the best-studied eukaryotic model system. We demonstrate detection of the known as well as new modifications, reveal partial modifications and unexpected communication between modification events, and determine the order of modification at several sites during ribosome biogenesis. Surprisingly, the method also provides information on a subset of other modifications. Hence, RiboMeth-seq enables a detailed evaluation of the importance of RNA modifications in the cells most sophisticated molecular machine. RiboMeth-seq can be adapted to other RNA classes, for example, mRNA, to reveal new biology involving RNA modifications.

Loss of N-terminal acetyltransferase A activity induces thermally unstable ribosomal proteins and increases their turnover in Saccharomyces cerevisiae.

Protein N-terminal (Nt) acetylation is one of the most abundant modifications in eukaryotes, covering ~50-80 % of the proteome, depending on species. Cells with defective Nt-acetylation display a wide array of phenotypes such as impaired growth, mating defects and increased stress sensitivity. However, the pleiotropic nature of these effects has hampered our understanding of the functional impact of protein Nt-acetylation. The main enzyme responsible for Nt-acetylation throughout the eukaryotic kingdom is the N-terminal acetyltransferase NatA. Here we employ a multi-dimensional proteomics approach to analyze Saccharomyces cerevisiae lacking NatA activity, which causes global proteome remodeling. Pulsed-SILAC experiments reveals that NatA-deficient strains consistently increase degradation of ribosomal proteins compared to wild type. Explaining this phenomenon, thermal proteome profiling uncovers decreased thermostability of ribosomes in NatA-knockouts. Our data are in agreement with a role for Nt-acetylation in promoting stability for parts of the proteome by enhancing the avidity of protein-protein interactions and folding.

Ribosomal RNA 2'-O-methylation dynamics impact cell fate decisions.

Translational regulation impacts both pluripotency maintenance and cell differentiation. To what degree the ribosome exerts control over this process remains unanswered. Accumulating evidence has demonstrated heterogeneity in ribosome composition in various organisms. 2'-O-methylation (2'-O-me) of rRNA represents an important source of heterogeneity, where site-specific alteration of methylation levels can modulate translation. Here, we examine changes in rRNA 2'-O-me during mouse brain development and tri-lineage differentiation of human embryonic stem cells (hESCs). We find distinct alterations between brain regions, as well as clear dynamics during cortex development and germ layer differentiation. We identify a methylation site impacting neuronal differentiation. Modulation of its methylation levels affects ribosome association of the fragile X mental retardation protein (FMRP) and is accompanied by an altered translation of WNT pathway-related mRNAs. Together, these data identify ribosome heterogeneity through rRNA 2'-O-me during early development and differentiation and suggest a direct role for ribosomes in regulating translation during cell fate acquisition.

Use of a Lariat Capping Ribozyme to Study Cap Function In Vivo.

A lariat cap is a naturally occurring substitute of a conventional mRNA cap and is found in a particular genomic setting in a few eukaryotic microorganisms. It is installed by the lariat capping ribozyme acting in cis. In principle, any RNA molecule in any organism can be equipped with a lariat cap in vivo when expressed downstream of a lariat capping ribozyme. Lariat capping is thus a versatile tool for studying the importance of the 5' end structure of RNA molecules. In this chapter, we present protocols to validate the presence of the lariat cap and measure the efficiency of in vivo cleavage by the lariat capping ribozyme.

The small Cajal body-specific RNA 15 (SCARNA15) directs p53 and redox homeostasis via selective splicing in cancer cells.

Small Cajal body-specific RNAs (scaRNAs) guide post-transcriptional modification of spliceosomal RNA and, while commonly altered in cancer, have poorly defined roles in tumorigenesis. Here, we uncover that SCARNA15 directs alternative splicing (AS) and stress adaptation in cancer cells. Specifically, we find that SCARNA15 guides critical pseudouridylation (Ψ) of U2 spliceosomal RNA to fine-tune AS of distinct transcripts enriched for chromatin and transcriptional regulators in malignant cells. This critically impacts the expression and function of the key tumor suppressors ATRX and p53. Significantly, SCARNA15 loss impairs p53-mediated redox homeostasis and hampers cancer cell survival, motility and anchorage-independent growth. In sum, these findings highlight an unanticipated role for SCARNA15 and Ψ in directing cancer-associated splicing programs.

The RNA helicase Dbp7 promotes domain V/VI compaction and stabilization of inter-domain interactions during early 60S assembly.

Early pre-60S ribosomal particles are poorly characterized, highly dynamic complexes that undergo extensive rRNA folding and compaction concomitant with assembly of ribosomal proteins and exchange of assembly factors. Pre-60S particles contain numerous RNA helicases, which are likely regulators of accurate and efficient formation of appropriate rRNA structures. Here we reveal binding of the RNA helicase Dbp7 to domain V/VI of early pre-60S particles in yeast and show that in the absence of this protein, dissociation of the Npa1 scaffolding complex, release of the snR190 folding chaperone, recruitment of the A3 cluster factors and binding of the ribosomal protein uL3 are impaired. uL3 is critical for formation of the peptidyltransferase center (PTC) and is responsible for stabilizing interactions between the 5' and 3' ends of the 25S, an essential pre-requisite for subsequent pre-60S maturation events. Highlighting the importance of pre-ribosome remodeling by Dbp7, our data suggest that in the absence of Dbp7 or its catalytic activity, early pre-ribosomal particles are targeted for degradation.

Regulation of translation by site-specific ribosomal RNA methylation.

Ribosomes are complex ribozymes that interpret genetic information by translating messenger RNA (mRNA) into proteins. Natural variation in ribosome composition has been documented in several organisms and can arise from several different sources. A key question is whether specific control over ribosome heterogeneity represents a mechanism by which translation can be regulated. We used RiboMeth-seq to demonstrate that differential 2'-O-methylation of ribosomal RNA (rRNA) represents a considerable source of ribosome heterogeneity in human cells, and that modification levels at distinct sites can change dynamically in response to upstream signaling pathways, such as MYC oncogene expression. Ablation of one prominent methylation resulted in altered translation of select mRNAs and corresponding changes in cellular phenotypes. Thus, differential rRNA 2'-O-methylation can give rise to ribosomes with specialized function. This suggests a broader mechanism where the specific regulation of rRNA modification patterns fine tunes translation.

Impaired Vitamin D Signaling in T Cells From a Family With Hereditary Vitamin D Resistant Rickets.

The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), mediates its immunomodulatory effects by binding to the vitamin D receptor (VDR). Here, we describe a new point mutation in the DNA-binding domain of the VDR and its consequences for 1,25(OH)2D3 signaling in T cells from heterozygous and homozygous carriers of the mutation. The mutation did not affect the overall structure or the ability of the VDR to bind 1,25(OH)2D3 and the retinoid X receptor. However, the subcellular localization of the VDR was strongly affected and the transcriptional activity was abolished by the mutation. In heterozygous carriers of the mutation, 1,25(OH)2D3-induced gene regulation was reduced by ~ 50% indicating that the expression level of wild-type VDR determines 1,25(OH)2D3 responsiveness in T cells. We show that vitamin D-mediated suppression of vitamin A-induced gene regulation depends on an intact ability of the VDR to bind DNA. Furthermore, we demonstrate that vitamin A inhibits 1,25(OH)2D3-induced translocation of the VDR to the nucleus and 1,25(OH)2D3-induced up-regulation of CYP24A1. Taken together, this study unravels novel aspects of vitamin D signaling and function of the VDR in human T cells.

Spatial-proteomics reveals phospho-signaling dynamics at subcellular resolution.

Dynamic change in subcellular localization of signaling proteins is a general concept that eukaryotic cells evolved for eliciting a coordinated response to stimuli. Mass spectrometry-based proteomics in combination with subcellular fractionation can provide comprehensive maps of spatio-temporal regulation of protein networks in cells, but involves laborious workflows that does not cover the phospho-proteome level. Here we present a high-throughput workflow based on sequential cell fractionation to profile the global proteome and phospho-proteome dynamics across six distinct subcellular fractions. We benchmark the workflow by studying spatio-temporal EGFR phospho-signaling dynamics in vitro in HeLa cells and in vivo in mouse tissues. Finally, we investigate the spatio-temporal stress signaling, revealing cellular relocation of ribosomal proteins in response to hypertonicity and muscle contraction. Proteomics data generated in this study can be explored through https://SpatialProteoDynamics.github.io .

Profiling of ribose methylations in ribosomal RNA from diffuse large B-cell lymphoma patients for evaluation of ribosomes as drug targets.

Cancer cells are addicted to ribosome biogenesis and high levels of translation. Thus, differential inhibition of cancer cells can be achieved by targeting aspects of ribosome biogenesis or ribosome function. Using RiboMeth-seq for profiling of the ∼112 2'-O-Me sites in human ribosomal RNA, we demonstrated pronounced hypomethylation at several sites in patient-derived diffuse large B-cell lymphoma (DLBCL) cell lines with a more severe perturbation in ABC-DLBCL compared to GBC-DLBCL. We extended our analysis to tumor samples from patients and demonstrated significant changes to the ribosomal modification pattern that appeared to consist of cell growth-related as well as tumor-specific changes. Sites of hypomethylation in patient samples are discussed as potential drug targets, using as an example a site in the small subunit (SSU-C1440) located in a ribosomal substructure that can be linked to DLBCL pathogenesis.