Loss of Ribosomal Protein Paralog Rpl22-like1 Blocks Lymphoid Development without Affecting Protein Synthesis.

Ribosomal proteins are thought to primarily facilitate biogenesis of the ribosome and its ability to synthesize protein. However, in this study, we show that Rpl22-like1 (Rpl22l1) regulates hematopoiesis without affecting ribosome biogenesis or bulk protein synthesis. Conditional loss of murine Rpl22l1 using stage or lineage-restricted Cre drivers impairs development of several hematopoietic lineages. Specifically, Tie2-Cre-mediated ablation of Rpl22l1 in hemogenic endothelium impairs the emergence of embryonic hematopoietic stem cells. Ablation of Rpl22l1 in late fetal liver progenitors impairs the development of B lineage progenitors at the pre-B stage and development of T cells at the CD44-CD25+ double-negative stage. In vivo labeling with O-propargyl-puromycin revealed that protein synthesis at the stages of arrest was not altered, indicating that the ribosome biogenesis and function were not generally compromised. The developmental arrest was associated with p53 activation, suggesting that the arrest may be p53-dependent. Indeed, development of both B and T lymphocytes was rescued by p53 deficiency. p53 induction was not accompanied by DNA damage as indicated by phospho-γH2AX induction or endoplasmic reticulum stress, as measured by phosphorylation of EIF2α, thereby excluding the known likely p53 inducers as causal. Finally, the developmental arrest of T cells was not rescued by elimination of the Rpl22l1 paralog, Rpl22, as we had previously found for the emergence of hematopoietic stem cells. This indicates that Rpl22 and Rpl22l1 play distinct and essential roles in supporting B and T cell development.

Disruption of Thrombocyte and T Lymphocyte Development by a Mutation in ARPC1B.

Regulation of the actin cytoskeleton is crucial for normal development and function of the immune system, as evidenced by the severe immune abnormalities exhibited by patients bearing inactivating mutations in the Wiskott-Aldrich syndrome protein (WASP), a key regulator of actin dynamics. WASP exerts its effects on actin dynamics through a multisubunit complex termed Arp2/3. Despite the critical role played by Arp2/3 as an effector of WASP-mediated control over actin polymerization, mutations in protein components of the Arp2/3 complex had not previously been identified as a cause of immunodeficiency. Here, we describe two brothers with hematopoietic and immunologic symptoms reminiscent of Wiskott-Aldrich syndrome (WAS). However, these patients lacked mutations in any of the genes previously associated with WAS. Whole-exome sequencing revealed a homozygous 2 bp deletion, n.c.G623DEL-TC (p.V208VfsX20), in Arp2/3 complex component ARPC1B that causes a frame shift resulting in premature termination. Modeling of the disease in zebrafish revealed that ARPC1B plays a critical role in supporting T cell and thrombocyte development. Moreover, the defects in development caused by ARPC1B loss could be rescued by the intact human ARPC1B ortholog, but not by the p.V208VfsX20 variant identified in the patients. Moreover, we found that the expression of ARPC1B is restricted to hematopoietic cells, potentially explaining why a mutation in ARPC1B has now been observed as a cause of WAS, whereas mutations in other, more widely expressed, components of the Arp2/3 complex have not been observed.

Reduced expression of the mouse ribosomal protein Rpl17 alters the diversity of mature ribosomes by enhancing production of shortened 5.8S rRNA.

Processing of rRNA during ribosome assembly can proceed through alternative pathways but it is unclear whether this could affect the structure of the ribosome. Here, we demonstrate that shortage of a ribosomal protein can change pre-rRNA processing in a way that over time alters ribosome diversity in the cell. Reducing the amount of Rpl17 in mouse cells led to stalled 60S subunit maturation, causing degradation of most of the synthesized precursors. A fraction of pre-60S subunits, however, were able to complete maturation, but with a 5'-truncated 5.8S rRNA, which we named 5.8SC. The 5' exoribonuclease Xrn2 is involved in the generation of both 5.8S(C) and the canonical long form of 5.8S rRNA. Ribosomes containing 5.8S(C) rRNA are present in various mouse and human cells and engage in translation. These findings uncover a previously undescribed form of mammalian 5.8S rRNA and demonstrate that perturbations in ribosome assembly can be a source of heterogeneity in mature ribosomes.

Regulatory Roles of Rpl22 in Hematopoiesis: An Old Dog with New Tricks.

Ribosomal proteins have long been known to serve critical roles in facilitating the biogenesis of the ribosome and its ability to synthesize proteins. However, evidence is emerging that suggests ribosomal proteins are also capable of performing tissue-restricted, regulatory functions that impact normal development and pathological conditions, including cancer. The challenge in studying such regulatory functions is that elimination of many ribosomal proteins also disrupts ribosome biogenesis and/or function. Thus, it is difficult to determine whether developmental abnormalities resulting from ablation of a ribosomal protein result from loss of core ribosome functions or from loss of the regulatory function of the ribosomal protein. Rpl22, a ribosomal protein component of the large 60S subunit, provides insight into this conundrum; Rpl22 is dispensable for both ribosome biogenesis and protein synthesis yet its ablation causes tissue-restricted disruptions in development. Here we review evidence supporting the regulatory functions of Rpl22 and other ribosomal proteins.

Two orthogonal cleavages separate subunit RNAs in mouse ribosome biogenesis.

Ribosome biogenesis is a dynamic multistep process, many features of which are still incompletely documented. Here, we show that changes in this pathway can be captured and annotated by means of a graphic set of pre-rRNA ratios, a technique we call Ratio Analysis of Multiple Precursors (RAMP). We find that knocking down a ribosome synthesis factor produces a characteristic RAMP profile that exhibits consistency across a range of depletion levels. This facilitates the inference of affected steps and simplifies comparative analysis. We applied RAMP to examine how endonucleolytic cleavages of the mouse pre-rRNA transcript in the internal transcribed spacer 1 (ITS1) are affected by depletion of factors required for maturation of the small ribosomal subunit (Rcl1, Fcf1/Utp24, Utp23) and the large subunit (Pes1, Nog1). The data suggest that completion of early maturation in a subunit triggers its release from the common pre-rRNA transcript by stimulating cleavage at the proximal site in ITS1. We also find that splitting of pre-rRNA in the 3' region of ITS1 is prevalent in adult mouse tissues and quiescent cells, as it is in human cells. We propose a model for subunit separation during mammalian ribosome synthesis and discuss its implications for understanding pre-rRNA processing pathways.

5'-end surveillance by Xrn2 acts as a shared mechanism for mammalian pre-rRNA maturation and decay.

Ribosome biogenesis requires multiple nuclease activities to process pre-rRNA transcripts into mature rRNA species and eliminate defective products of transcription and processing. We find that in mammalian cells, the 5' exonuclease Xrn2 plays a major role in both maturation of rRNA and degradation of a variety of discarded pre-rRNA species. Precursors of 5.8S and 28S rRNAs containing 5' extensions accumulate in mouse cells after siRNA-mediated knockdown of Xrn2, indicating similarity in the 5'-end maturation mechanisms between mammals and yeast. Strikingly, degradation of many aberrant pre-rRNA species, attributed mainly to 3' exonucleases in yeast studies, occurs 5' to 3' in mammalian cells and is mediated by Xrn2. Furthermore, depletion of Xrn2 reveals pre-rRNAs derived by cleavage events that deviate from the main processing pathway. We propose that probing of pre-rRNA maturation intermediates by exonucleases serves the dual function of generating mature rRNAs and suppressing suboptimal processing paths during ribosome assembly.

Ribosomal protein control of hematopoietic stem cell transformation through direct, non-canonical regulation of metabolism.

We report here that expression of the ribosomal protein, RPL22, is frequently reduced in human myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML); reduced RPL22 expression is associated with worse outcomes. Mice null for Rpl22 display characteristics of an MDS-like syndrome and develop leukemia at an accelerated rate. Rpl22-deficient mice also display enhanced hematopoietic stem cell (HSC) self-renewal and obstructed differentiation potential, which arises not from reduced protein synthesis but from increased expression of the Rpl22 target, ALOX12, an upstream regulator of fatty acid oxidation (FAO). The increased FAO mediated by Rpl22-deficiency also persists in leukemia cells and promotes their survival. Altogether, these findings reveal that Rpl22 insufficiency enhances the leukemia potential of HSC via non-canonical de-repression of its target, ALOX12, which enhances FAO, a process that may serve as a therapeutic vulnerability of Rpl22 low MDS and AML leukemia cells. Highlights: RPL22 insufficiency is observed in MDS/AML and is associated with reduced survivalRpl22-deficiency produces an MDS-like syndrome and facilitates leukemogenesisRpl22-deficiency does not impair global protein synthesis by HSCRpl22 controls leukemia cell survival by non-canonical regulation of lipid oxidation eTOC: Rpl22 controls the function and transformation potential of hematopoietic stem cells through effects on ALOX12 expression, a regulator of fatty acid oxidation.

Polyadenylation and degradation of incomplete RNA polymerase I transcripts in mammalian cells.

Most transcripts in growing cells are ribosomal RNA precursors (pre-rRNA). Here, we show that in mammals, aberrant pre-rRNA transcripts generated by RNA polymerase I (Pol I) are polyadenylated and accumulate markedly after treatment with low concentrations of actinomycin D (ActD), which blocks the synthesis of full-length rRNA. The poly(A) polymerase-associated domain-containing protein 5 is required for polyadenylation, whereas the exosome is partly responsible for the degradation of the short aberrant transcripts. Thus, polyadenylation functions in the quality control of Pol I transcription in metazoan cells. The impact of excessive aberrant RNAs on the degradation machinery is an unrecognized mechanism that might contribute to biological properties of ActD.

Antimicrobial and Aging Properties of Ag-, Ag/Cu-, and Ag Cluster-Doped Amorphous Carbon Coatings Produced by Magnetron Sputtering for Space Applications.

Inside a spacecraft, the temperature and humidity, suitable for the human crew onboard, also creates an ideal breeding environment for the proliferation of bacteria and fungi; this can present a hazard to human health and create issues for the safe running of equipment. To address this issue, wear-resistant antimicrobial thin films prepared by magnetron sputtering were developed, with the aim to coat key internal components within spacecrafts. Silver and copper are among the most studied active bactericidal materials, thus this work investigated the antibacterial properties of amorphous carbon coatings, doped with either silver, silver and copper, or with silver clusters. The longevity of these antimicrobial coatings, which is heavily influenced by metal diffusion within the coating, was also investigated. With a conventional approach, amorphous carbon coatings were prepared by cosputtering, to generate coatings that contained a range of silver and copper concentrations. In addition, coatings containing silver clusters were prepared using a separate cluster source to better control the metal particle size distribution in the amorphous carbon matrix. The particle size distributions were characterized by grazing-incidence small-angle X-ray scattering (GISAXS). Antibacterial tests were performed under both terrestrial gravity and microgravity conditions, to simulate the condition in space. Results show that although silver-doped coatings possess extremely high levels of antimicrobial activity, silver cluster-doped coatings are equally effective, while being more long-lived, despite containing a lower absolute silver concentration.

Early transcriptional arrest at Escherichia coli rplN and ompX promoters.

Bacterial transcription elongation factors GreA and GreB stimulate the intrinsic RNase activity of RNA polymerase (RNAP), thus helping the enzyme to read through pausing and arresting sites on DNA. Gre factors also accelerate RNAP transition from initiation to elongation. Here, we characterized the molecular mechanism by which Gre factors facilitate transcription at two Escherichia coli promoters, PrplN and PompX, that require GreA for optimal in vivo activity. Using in vitro transcription assays, KMnO(4) footprinting, and Fe(2+)-induced hydroxyl radical mapping, we show that during transcription initiation at PrplN and PompX in the absence of Gre factors, RNAP falls into a condition of promoter-proximal transcriptional arrest that prevents production of full-length transcripts both in vitro and in vivo. Arrest occurs when RNAP synthesizes 9-14-nucleotide-long transcripts and backtracks by 5-7 (PrplN) or 2-4 (PompX) nucleotides. Initiation factor sigma(70) contributes to the formation of arrested complexes at both promoters. The signal for promoter-proximal arrest at PrplN is bipartite and requires two elements: the extended -10 promoter element and the initial transcribed region from positions +2 to +6. GreA and GreB prevent arrest at PrplN and PompX by inducing cleavage of the 3'-proximal backtracked portion of RNA at the onset of arrested complex formation and stimulate productive transcription by allowing RNAP to elongate the 5'-proximal transcript cleavage products in the presence of substrates. We propose that promoter-proximal arrest is a common feature of many bacterial promoters and may represent an important physiological target of regulation by transcript cleavage factors.

Ribosomal Proteins Rpl22 and Rpl22l1 Control Morphogenesis by Regulating Pre-mRNA Splicing.

Most ribosomal proteins (RP) are regarded as essential, static components that contribute only to ribosome biogenesis and protein synthesis. However, emerging evidence suggests that RNA-binding RP are dynamic and can influence cellular processes by performing "extraribosomal," regulatory functions involving binding to select critical target mRNAs. We report here that the RP, Rpl22, and its highly homologous paralog Rpl22-Like1 (Rpl22l1 or Like1) play critical, extraribosomal roles in embryogenesis. Indeed, they antagonistically control morphogenesis through developmentally regulated localization to the nucleus, where they modulate splicing of the pre-mRNA encoding smad2, an essential transcriptional effector of Nodal/TGF-ß signaling. During gastrulation, Rpl22 binds to intronic sequences of smad2 pre-mRNA and induces exon 9 skipping in cooperation with hnRNP-A1. This action is opposed by its paralog, Like1, which promotes exon 9 inclusion in the mature transcript. The nuclear roles of these RP in controlling morphogenesis represent a fundamentally different and paradigm-shifting mode of action for RP.

Quantitative Northern Blot Analysis of Mammalian rRNA Processing.

Assembly of eukaryotic ribosomes is an elaborate biosynthetic process that begins in the nucleolus and requires hundreds of cellular factors. Analysis of rRNA processing has been instrumental for studying the mechanisms of ribosome biogenesis and effects of stress conditions on the molecular milieu of the nucleolus. Here, we describe the quantitative analysis of the steady-state levels of rRNA precursors, applicable to studies in mammalian cells and other organisms. We include protocols for gel electrophoresis and northern blotting of rRNA precursors using procedures optimized for the large size of these RNAs. We also describe the ratio analysis of multiple precursors, a technique that facilitates the accurate assessment of changes in the efficiency of individual pre-rRNA processing steps.

LipL41, a hemin binding protein from Leptospira santarosai serovar Shermani.

Leptospirosis is one of the most widespread zoonotic diseases in the world. It is caused by the pathogen Leptospira that results in multiple-organ failure, in particular of the kidney. Outer membrane lipoprotein is the suspected virulence factor of Leptospira. In Leptospira spp LipL41 is one major lipoprotein and is highly conserved. Previous study suggests that LipL41 bears hemin-binding ability and might play a possible role in iron regulation and storage. However, the characterization of hemin-binding ability of LipL41 is still unclear. Here the hemin-binding ability of LipL41 was examined, yielding a K d = 0.59 ± 0.14 µM. Two possible heme regulatory motifs (HRMs), C[P/S], were found in LipL41 at (140)Cys-Ser and (220)Cys-Pro. The mutation study indicates that Cys140 and Cys220 might be cooperatively involved in hemin binding. A supramolecular assembly of LipL41 was determined by transmission electron microscopy. The LipL41 oligomer consists of 36 molecules and folds as a double-layered particle. At the C-terminus of LipL41, there are two tetratricopeptide repeats (TPRs), which might be involved in the protein-protein interaction of the supramolecular assembly.

Mammalian DEAD box protein Ddx51 acts in 3' end maturation of 28S rRNA by promoting the release of U8 snoRNA.

Biogenesis of eukaryotic ribosomes requires a number of RNA helicases that drive molecular rearrangements at various points of the assembly pathway. While many ribosome synthesis factors are conserved among all eukaryotes, certain features of ribosome maturation, such as U8 snoRNA-assisted processing of the 5.8S and 28S rRNA precursors, are observed only in metazoan cells. Here, we identify the mammalian DEAD box helicase family member Ddx51 as a novel ribosome synthesis factor and an interacting partner of the nucleolar GTP-binding protein Nog1. Unlike any previously studied yeast helicases, Ddx51 is required for the formation of the 3' end of 28S rRNA. Ddx51 binds to pre-60S subunit complexes and promotes displacement of U8 snoRNA from pre-rRNA, which is necessary for the removal of the 3' external transcribed spacer from 28S rRNA and productive downstream processing. These data demonstrate the emergence of a novel factor that facilitates a pre-rRNA processing event specific for higher eukaryotes.