The RNA-binding landscape of HAX1 protein indicates its involvement in ribosome biogenesis and translation.

HAX1 is a human protein with no known homologues or structural domains, mutations in which cause severe congenital neutropenia through mechanisms that are poorly understood. Previous studies reported RNA-binding capacity of HAX1, but the role of this binding in physiology and pathology remains unexplained. Here we report transcriptome-wide characterization of HAX1 RNA targets using RIP-seq and CRAC, indicating that HAX1 binds transcripts involved in ribosome biogenesis and rRNA processing. Using CRISPR knockouts we find that RNA targets of HAX1 partially overlap with transcripts downregulated in HAX1 KO, implying a role in mRNA stabilization. Gene ontology analysis demonstrated that genes differentially expressed in HAX1 KO (including genes involved in ribosome biogenesis and translation) are also enriched in a subset of genes whose expression correlates with HAX1 expression in four analyzed neoplasms. Functional connection to ribosome biogenesis was also demonstrated by gradient sedimentation ribosome profiles, which revealed differences in the small subunit:monosome ratio in HAX1 WT/KO. We speculate that changes in HAX1 expression may be important for the etiology of HAX1-linked diseases through dysregulation of translation.

Deficiency of the Ribosomal Protein uL5 Leads to Significant Rearrangements of the Transcriptional and Translational Landscapes in Mammalian Cells

Protein uL5 (formerly called L11) is an integral component of the large (60S) subunit of the human ribosome, and its deficiency in cells leads to the impaired biogenesis of 60S subunits. Using RNA interference, we reduced the level of uL5 in HEK293T cells by three times, which caused an almost proportional decrease in the content of the fraction corresponding to 80S ribosomes, without a noticeable diminution in the level of polysomes. By RNA sequencing of uL5-deficient and control cell samples, which were those of total mRNA and mRNA from the polysome fraction, we identified hundreds of differentially expressed genes (DEGs) at the transcriptome and translatome levels and revealed dozens of genes with altered translational efficiency (GATEs). Transcriptionally up-regulated DEGs were mainly associated with rRNA processing, pre-mRNA splicing, translation and DNA repair, while down-regulated DEGs were genes of membrane proteins; the type of regulation depended on the GC content in the 3′ untranslated regions of DEG mRNAs. The belonging of GATEs to up-regulated and down-regulated ones was determined by the coding sequence length of their mRNAs. Our findings suggest that the effects observed in uL5-deficient cells result from an insufficiency of translationally active ribosomes caused by a deficiency of 60S subunits.

RNase III-mediated processing of a trans-acting bacterial sRNA and its cis-encoded antagonist

Bacterial small RNAs (sRNAs) are important post-transcriptional regulators in stress responses and virulence. They can be derived from an expanding list of genomic contexts, such as processing from parental transcripts by RNase E. The role of RNase III in sRNA biogenesis is less well understood despite its well-known roles in rRNA processing, RNA decay, and cleavage of sRNA-mRNA duplexes. Here, we show that RNase III processes a pair of cis-encoded sRNAs (CJnc190 and CJnc180) of the foodborne pathogen Campylobacter jejuni. While CJnc180 processing by RNase III requires CJnc190, In contrast, RNase III processes CJnc190 independent of CJnc180 via cleavage of an intramolecular duplex. We also show that CJnc190 directly represses translation of the colonization factor PtmG by targeting a G-rich ribosome binding site, and uncover that CJnc180 is a cis-acting antagonist of CJnc190, indirectly affecting ptmG regulation. Our study highlights a role for RNase III in sRNA biogenesis and adds cis-encoded RNAs to the expanding diversity of transcripts that antagonize bacterial sRNAs.

hNOP2/NSUN1 Regulates Ribosome Biogenesis through Stabilization of snoRNP Complexes and Cytosine-5 Methylation of 28S rRNA.

5-Methylcytosine (m5C) is a base modification broadly found on a variety of RNAs in the human transcriptome. In eukaryotes m5C is catalyzed by enzymes of the NSUN family, which is composed of seven members in humans (NSUN1-7). NOP2/NSUN1 has been mostly characterized in budding yeast as an essential ribosome biogenesis factor required for the deposition of m5C on the 25S rRNA. Although human NOP2/NSUN1 has been known to be an oncogene overexpressed in several types of cancer, its functions remain poorly characterized. To define the roles of human NOP2/NSUN1, we used an miCLIP-seq approach to identify its RNA substrates. Our analysis reveals that vault RNA 1.2 and rRNA are NOP2/NSUN1-specific methylated targets and we further confirm by bisulfite sequencing that NOP2/NSUN1 is responsible for the deposition of m5C at residue 4447 on the 28S rRNA. Depletion of NOP2/NSUN1 impairs cell proliferation, rRNA processing and 60S ribosome biogenesis. Additionally, we find that NOP2/NSUN1 binds to the 5′ETS region of the pre-rRNA transcript and regulates pre-rRNA processing in part through non-catalytic complex formation with box C/D snoRNAs. Our study identifies for the first time the RNA substrates of human NOP2/NSUN1 and reveals additional functions in rRNA processing beyond catalyzing m5C base modification.

EXOSC10/Rrp6 is essential for the eight-cell embryo/morula transition

The mouse 3′-5′ exoribonuclease EXOSC10/Rrp6 is required for rRNA processing, gametogenesis, brain development, erythropoiesis and blood cell enhancer function. The human ortholog is essential for mitosis in cancer cells and its enzymatic activity is inhibited by the anti-cancer drug 5-fluorouracil. Little is known, however, about the role of Exosc10 during embryo development and organogenesis. We generated an Exosc10 knockout model and find that Exosc10-/- mice show an embryonic lethal phenotype. We demonstrate that Exosc10 maternal mRNA is present in mutant oocytes and that the gene is expressed during early embryogenesis. Furthermore, we observe that EXOSC10 localizes to the periphery of nucleolar precursor bodies and nucleoli in blastomeres, which is consistent with the protein's role in rRNA processing. Finally, we infer from genotyping data obtained with samples harvested at embryonic days e7.5, e6.5 and e4.5 and embryos cultured in vitro that Exosc10-/- mutants arrest at the eight-cell embryo/morula transition. Our results demonstrate a novel essential role for Exosc10 during early embryogenesis, and they are consistent with earlier work showing that impaired ribosome biogenesis causes a developmental arrest at the morula stage.

The nucleolar DExD/H protein Hel66 is involved in ribosome biogenesis in Trypanosoma brucei

The biosynthesis of ribosomes is a complex cellular process involving ribosomal RNA, ribosomal proteins and several further trans-acting factors. DExD/H box proteins constitute the largest family of trans-acting protein factors involved in this process. Several members of this protein family have been directly implicated in ribosome biogenesis in yeast. In trypanosomes, ribosome biogenesis differs in several features from the process described in yeast. Here, we have identified the DExD/H box helicase Hel66 as being involved in ribosome biogenesis. The protein is unique to Kinetoplastida, localises to the nucleolus and its depletion via RNAi caused a severe growth defect. Loss of the protein resulted in a decrease of global translation and accumulation of rRNA processing intermediates for both the small and large ribosomal subunits. Only a few factors involved in trypanosome rRNA biogenesis have been described so far and our findings contribute to gaining a more comprehensive picture of this essential process.

Blocking a dead-end assembly pathway creates a point of regulation for the DEAD-box ATPase Has1 and prevents platform misassembly

Assembly of ribosomal subunits occurs via parallel pathways, which accelerate the process and render it more robust. Nonetheless, in vitro analyses have also demonstrated that some assembly pathways are dead-ends, presumably due to rRNA misfolding. If and how these non-productive pathways are avoided during assembly in vivo remains unknown. Here we use a combination of biochemical, genetic, proteomic and structural analyses to demonstrate a role for assembly factors in biasing the folding landscape away from non-productive intermediates. By binding Rrp36, Rrp5 is prevented from forming a premature interaction with the platform, which leads to a dead-end intermediate, and a misassembled platform that is functionally defective. The DEAD-box ATPase Has1 separates Rrp5 and Rrp36, allowing Rrp5 to reposition to the platform, thereby promoting ribosome assembly and enabling rRNA processing. Thus, Rrp36 establishes an ATP-dependent regulatory point that ensures correct platform assembly by opening a new folding channel that avoids funnels to misfolding.

Ancestral protein topologies draw the rooted bacterial tree of life

Many experimental and predicted observations remain unanswered in the current proposed trees of life (ToL). Also, the current trend in reporting phylogenetic data is based in mixing together the information of dozens of genomes or entire conserved proteins. In this work, we consider the modularity of protein evolution and, using only two domains with duplicated ancestral topologies from a single, universal primordial protein corresponding to the RNA binding regions of contemporary bacterial glycyl tRNA synthetase (bacGlyRS), archaeal CCA adding enzyme (arch-CCAadd) and eukaryotic rRNA processing enzyme (euk-rRNA), we propose a rooted bacterial ToL that agrees with several previous observations unaccounted by the available trees.