Cell surface-bound La protein regulates the cell fusion stage of osteoclastogenesis.

Multinucleated osteoclasts, essential for skeletal remodeling in health and disease, are formed by the fusion of osteoclast precursors, where each fusion event raises their bone-resorbing activity. Here we show that the nuclear RNA chaperone, La protein has an additional function as an osteoclast fusion regulator. Monocyte-to-osteoclast differentiation starts with a drastic decrease in La levels. As fusion begins, La reappears as a low molecular weight species at the osteoclast surface, where it promotes fusion. La's role in promoting osteoclast fusion is independent of canonical La-RNA interactions and involves direct interactions between La and Annexin A5, which anchors La to transiently exposed phosphatidylserine at the surface of fusing osteoclasts. Disappearance of cell-surface La, and the return of full length La to the nuclei of mature, multinucleated osteoclasts, acts as an off switch of their fusion activity. Targeting surface La in a novel explant model of fibrous dysplasia inhibits excessive osteoclast formation characteristic of this disease, highlighting La's potential as a therapeutic target.

The La and related RNA-binding proteins (LARPs): structures, functions, and evolving perspectives.

La was first identified as a polypeptide component of ribonucleic protein complexes targeted by antibodies in autoimmune patients and is now known to be a eukaryote cell-ubiquitous protein. Structure and function studies have shown that La binds to a common terminal motif, UUU-3'-OH, of nascent RNA polymerase III (RNAP III) transcripts and protects them from exonucleolytic decay. For precursor-tRNAs, the most diverse and abundant of these transcripts, La also functions as an RNA chaperone that helps to prevent their misfolding. Related to this, we review evidence that suggests that La and its link to RNAP III were significant in the great expansions of the tRNAomes that occurred in eukaryotes. Four families of La-related proteins (LARPs) emerged during eukaryotic evolution with specialized functions. We provide an overview of the high-resolution structural biology of La and LARPs. LARP7 family members most closely resemble La but function with a single RNAP III nuclear transcript, 7SK, or telomerase RNA. A cytoplasmic isoform of La protein as well as LARPs 6, 4, and 1 function in mRNA metabolism and translation in distinct but similar ways, sometimes with the poly(A)-binding protein, and in some cases by direct binding to poly(A)-RNA. New structures of LARP domains, some complexed with RNA, provide novel insights into the functional versatility of these proteins. We also consider LARPs in relation to ancestral La protein and potential retention of links to specific RNA-related pathways. One such link may be tRNA surveillance and codon usage by LARP-associated mRNAs. WIREs RNA 2017, 8:e1430. doi: 10.1002/wrna.1430 For further resources related to this article, please visit the WIREs website.

LARP4 Is Regulated by Tumor Necrosis Factor Alpha in a Tristetraprolin-Dependent Manner.

LARP4 is a protein with unknown function that independently binds to poly(A) RNA, RACK1, and the poly(A)-binding protein (PABPC1). Here, we report on its regulation. We found a conserved AU-rich element (ARE) in the human LARP4 mRNA 3' untranslated region (UTR). This ARE, but not its antisense version or a point-mutated version, significantly decreased the stability of ß-globin reporter mRNA. We found that overexpression of tristetraprolin (TTP), but not its RNA binding mutant or the other ARE-binding proteins tested, decreased cellular LARP4 levels. RNA coimmunoprecipitation showed that TTP specifically associated with LARP4 mRNA in vivo. Consistent with this, mouse LARP4 accumulated to higher levels in TTP gene knockout (KO) cells than in control cells. Stimulation of WT cells with tumor necrosis factor alpha (TNF-α), which rapidly induces TTP, robustly decreased LARP4 with a coincident time course but had no such effect on LARP4B or La protein or on LARP4 in the TTP KO cells. The TNF-α-induced TTP pulse was followed by a transient decrease in LARP4 mRNA that was quickly followed by a subsequent transient decrease in LARP4 protein. Involvement of LARP4 as a target of TNF-α-TTP regulation provides a clue as to how its functional activity may be used in a physiologic pathway.

Defective i6A37 modification of mitochondrial and cytosolic tRNAs results from pathogenic mutations in TRIT1 and its substrate tRNA.

Identifying the genetic basis for mitochondrial diseases is technically challenging given the size of the mitochondrial proteome and the heterogeneity of disease presentations. Using next-generation exome sequencing, we identified in a patient with severe combined mitochondrial respiratory chain defects and corresponding perturbation in mitochondrial protein synthesis, a homozygous p.Arg323Gln mutation in TRIT1. This gene encodes human tRNA isopentenyltransferase, which is responsible for i6A37 modification of the anticodon loops of a small subset of cytosolic and mitochondrial tRNAs. Deficiency of i6A37 was previously shown in yeast to decrease translational efficiency and fidelity in a codon-specific manner. Modelling of the p.Arg323Gln mutation on the co-crystal structure of the homologous yeast isopentenyltransferase bound to a substrate tRNA, indicates that it is one of a series of adjacent basic side chains that interact with the tRNA backbone of the anticodon stem, somewhat removed from the catalytic center. We show that patient cells bearing the p.Arg323Gln TRIT1 mutation are severely deficient in i6A37 in both cytosolic and mitochondrial tRNAs. Complete complementation of the i6A37 deficiency of both cytosolic and mitochondrial tRNAs was achieved by transduction of patient fibroblasts with wild-type TRIT1. Moreover, we show that a previously-reported pathogenic m.7480A>G mt-tRNASer(UCN) mutation in the anticodon loop sequence A36A37A38 recognised by TRIT1 causes a loss of i6A37 modification. These data demonstrate that deficiencies of i6A37 tRNA modification should be considered a potential mechanism of human disease caused by both nuclear gene and mitochondrial DNA mutations while providing insight into the structure and function of TRIT1 in the modification of cytosolic and mitochondrial tRNAs.

Human cells have a limited set of tRNA anticodon loop substrates of the tRNA isopentenyltransferase TRIT1 tumor suppressor.

Human TRIT1 is a tRNA isopentenyltransferase (IPTase) homologue of Escherichia coli MiaA, Saccharomyces cerevisiae Mod5, Schizosaccharomyces pombe Tit1, and Caenorhabditis elegans GRO-1 that adds isopentenyl groups to adenosine 37 (i6A37) of substrate tRNAs. Prior studies indicate that i6A37 increases translation fidelity and efficiency in codon-specific ways. TRIT1 is a tumor suppressor whose mutant alleles are associated with cancer progression. We report the systematic identification of i6A37-containing tRNAs in a higher eukaryote, performed using small interfering RNA knockdown and other methods to examine TRIT1 activity in HeLa cells. Although several potential substrates contained the IPTase recognition sequence A36A37A38 in the anticodon loop, only tRNA(Ser)AGA, tRNA(Ser)CGA, tRNA(Ser)UGA, and selenocysteine tRNA with UCA (tRNA([Ser]Sec)UCA) contained i6A37. This subset is a significantly more restricted than that for two distant yeasts (S. cerevisiae and S. pombe), the only other organisms comprehensively examined. Unlike the fully i6A37-modified tRNAs for Ser, tRNA([Ser]Sec)UCA is partially (∼40%) modified. Exogenous selenium and other treatments that decreased the i6A37 content of tRNA([Ser]Sec)UCA led to increased levels of the tRNA([Ser]Sec)UCA. Of the human mitochondrion (mt)-encoded tRNAs with A36A37A38, only mt tRNAs tRNA(Ser)UGA and tRNA(Trp)UCA contained detectable i6A37. Moreover, while tRNA(Ser) levels were unaffected by TRIT1 knockdown, the tRNA([Ser]Sec)UCA level was increased and the mt tRNA(Ser)UGA level was decreased, suggesting that TRIT1 may control the levels of some tRNAs as well as their specific activity.

Both endonucleolytic and exonucleolytic cleavage mediate ITS1 removal during human ribosomal RNA processing.

Human ribosome production is up-regulated during tumorogenesis and is defective in many genetic diseases (ribosomopathies). We have undertaken a detailed analysis of human precursor ribosomal RNA (pre-rRNA) processing because surprisingly little is known about this important pathway. Processing in internal transcribed spacer 1 (ITS1) is a key step that separates the rRNA components of the large and small ribosomal subunits. We report that this was initiated by endonuclease cleavage, which required large subunit biogenesis factors. This was followed by 3' to 5' exonucleolytic processing by RRP6 and the exosome, an enzyme complex not previously linked to ITS1 removal. In contrast, RNA interference-mediated knockdown of the endoribonuclease MRP did not result in a clear defect in ITS1 processing. Despite the apparently high evolutionary conservation of the pre-rRNA processing pathway and ribosome synthesis factors, each of these features of human ITS1 processing is distinct from those in budding yeast. These results also provide significant insight into the links between ribosomopathies and ribosome production in human cells.

Viperin mRNA is a novel target for the human RNase MRP/RNase P endoribonuclease.

RNase MRP is a conserved endoribonuclease, in humans consisting of a 267-nucleotide RNA associated with 7-10 proteins. Mutations in its RNA component lead to several autosomal recessive skeletal dysplasias, including cartilage-hair hypoplasia (CHH). Because the known substrates of mammalian RNase MRP, pre-ribosomal RNA, and RNA involved in mitochondrial DNA replication are not likely involved in CHH, we analyzed the effects of RNase MRP (and the structurally related RNase P) depletion on mRNAs using DNA microarrays. We confirmed the upregulation of the interferon-inducible viperin mRNA by RNAi experiments and this appeared to be independent of the interferon response. We detected two cleavage sites for RNase MRP/RNase P in the coding sequence of viperin mRNA. This is the first study providing direct evidence for the cleavage of a mRNA by RNase MRP/RNase P in human cells. Implications for the involvement in the pathophysiology of CHH are discussed.

RNase MRP and disease.

The human RNase MRP complex consists of a catalytic RNA and several protein components. RNase MRP is a ubiquitously expressed eukaryotic endoribonuclease that cleaves various RNAs, including ribosomal, messenger, and mitochondrial RNAs, in a highly specific fashion. In several autoimmune diseases autoantibodies targeting RNase MRP have been found. These so-called anti-Th/To autoantibodies, which most frequently can be detected in the sera of scleroderma patients, are directed to several protein components of the RNase MRP and the evolutionarily related RNase P complex. It is not yet known whether the anti-Th/To immune response is an epiphenomenon or whether these autoantibodies play a role in the pathophysiology of the disease. The gene encoding the RNase MRP RNA was the first nuclear non-coding RNA gene demonstrated to be associated with a genetic disease. Mutations in this gene are causing the highly pleiotropic disease cartilage-hair hypoplasia (CHH). CHH patients are characterized by a short stature, hypoplastic hair, and short limbs. In addition, they show a predisposition to lymphomas and other cancers and suffer from defective T-cell immunity. Since the identification of the first CHH-associated mutations in 2001, many distinct mutations have been found in different patients. These mutations either affect the structure of the RNase MRP RNA or are located in the promoter region and reduce the expression levels. In this review article we will, after describing the biochemical aspects of RNase MRP, discuss the targeting of RNase MRP in autoimmunity and the role of mutations in the RNase MRP RNA gene in CHH.

Cartilage-hair hypoplasia-associated mutations in the RNase MRP P3 domain affect RNA folding and ribonucleoprotein assembly.

Cartilage-hair hypoplasia (CHH) is caused by mutations in the gene encoding the RNA component of RNase MRP. Currently it is unknown how these mutations affect the function of this endoribonuclease. In this study we investigated the effect of mutations in the P3 domain on protein binding and RNA folding. Our data demonstrate that a number of P3 nucleotide substitutions reduced the efficiency of its interaction with Rpp25 and Rpp20, two protein subunits binding as a heterodimer to this domain. The CHH-associated 40G>A substitution, as well as the replacement of residue 47, almost completely abrogated Rpp25 and Rpp20 binding in different assays. Also other CHH-associated P3 mutations reduced the efficiency by which the RNase MRP RNA is bound by Rpp25-Rpp20. These data demonstrate that the most important residues for binding of the Rpp25-Rpp20 dimer reside in the apical stem-loop of the P3 domain. Structural analyses by NMR not only showed that this loop may adopt a pseudo-triloop structure, but also demonstrated that the 40G>A substitution alters the folding of this part of the P3 domain. Our data are the first to provide insight into the molecular mechanism by which CHH-associated mutations affect the function of RNase MRP.