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Dive into the research topics where Richard J. Maraia is active.

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Featured researches published by Richard J. Maraia.


Cell | 2006

A Role for TFIIIC Transcription Factor Complex in Genome Organization

Ken-ichi Noma; Hugh P. Cam; Richard J. Maraia; Shiv I. S. Grewal

Eukaryotic genome complexity necessitates boundary and insulator elements to partition genomic content into distinct domains. We show that inverted repeat (IR) boundary elements flanking the fission yeast mating-type heterochromatin domain contain B-box sequences, which prevent heterochromatin from spreading into neighboring euchromatic regions by recruiting transcription factor TFIIIC complex without RNA polymerase III (Pol III). Genome-wide analysis reveals TFIIIC with Pol III at all tRNA genes, many of which cluster at pericentromeric heterochromatin domain boundaries. However, a single tRNA(phe) gene with modest TFIIIC enrichment is insufficient to serve as boundary and requires RNAi-associated element to restrain heterochromatin spreading. Remarkably, we found TFIIIC localization without Pol III at many sites located between divergent promoters. These sites appear to act as chromosome-organizing clamps by tethering distant loci to the nuclear periphery, at which TFIIIC is concentrated into several distinct bodies. Our analyses uncover a general genome organization mechanism involving conserved TFIIIC complex.


Molecular and Cellular Biology | 2001

Recognition of nascent RNA by the human La antigen: conserved and divergent features of structure and function.

Richard J. Maraia; Robert V. Intine

La is a conserved RNA-binding phosphoprotein that interacts with a large variety of ligands. The most ubiquitous function of La is association with newly synthesized RNA polymerase (Pol) III transcripts via their common UUU-OH 39 termini and stabilization of these against exonucleolytic digestion. Accumulating evidence also indicates an activity for La in internal ribosome entry site-mediated translation in mammalian cells and in the metabolism of a subset of 39-processed snRNA intermediates that end in uridylates but are synthesized by Pol II. The most highly conserved region of La resides in the N-terminal domain (NTD), and this appears to mediate highaffinity UUU-OH recognition. As critically reviewed here by comparison to a consensus core RNA recognition motif (RRM) structure, the NTD can be modeled into a pair of tandem RRMs. In addition to the conserved NTD, human La (hLa) protein contains a C-terminal domain (CTD) that harbors a third RRM and a potential Walker A motif that appears to recognize the 59-ppp ends of nascent RNAs. The resulting bipartite mode of RNA binding can account for previously unexplained observations and may underlie a unifying principle of La function. While a role for hLa in transcription remains controversial, its presence in a Pol III holoenzyme suggests a role reminiscent of the CTD of Pol II, as an integrator of transcriptional and posttranscriptional activities that include 59- and 39-RNA metabolism. Evidence that the 59-end-RNA recognition activity of hLa can be modulated by phosphorylation provides mechanistic insight into the signal transduction


Molecular and Cellular Biology | 1998

5′ Processing of tRNA Precursors Can Be Modulated by the Human La Antigen Phosphoprotein

Hao Fan; John L. Goodier; Joel R. Chamberlain; David R. Engelke; Richard J. Maraia

ABSTRACT Eukaryotic precursor (pre)-tRNAs are processed at both ends prior to maturation. Pre-tRNAs and other nascent transcripts synthesized by RNA polymerase III are bound at their 3′ ends at the sequence motif UUUOH [3′ oligo(U)] by the La antigen, a conserved phosphoprotein whose role in RNA processing has been associated previously with 3′-end maturation only. We show that in addition to its role in tRNA 3′-end maturation, human La protein can also modulate 5′ processing of pre-tRNAs. Both the La antigen’s N-terminal RNA-binding domain and its C-terminal basic region are required for attenuation of pre-tRNA 5′ processing. RNA binding and nuclease protection assays with a variety of pre-tRNA substrates and mutant La proteins indicate that 5′ protection is a highly selective activity of La. This activity is dependent on 3′ oligo(U) in the pre-tRNA for interaction with the N-terminal RNA binding domain of La and interaction of the C-terminal basic region of La with the 5′ triphosphate end of nascent pre-tRNA. Phosphorylation of La is known to occur on serine 366, adjacent to the C-terminal basic region. We show that this modification interferes with the La antigen’s ability to protect pre-tRNAi Met from 5′ processing either by HeLa extract or purified RNase P but that it does not affect interaction with the 3′ end of pre-tRNA. These findings provide the first evidence to indicate that tRNA 5′-end maturation may be regulated in eukaryotes. Implications of triphosphate recognition is discussed as is a role for La phosphoprotein in controlling transcriptional and posttranscriptional events in the biogenesis of polymerase III transcripts.


Cell | 1997

Phosphorylation of the human La antigen on serine 366 can regulate recycling of RNA polymerase III transcription complexes.

Hao Fan; Amy L. Sakulich; John L. Goodier; Xiaolong Zhang; Jun Qin; Richard J. Maraia

The human La antigen is an RNA-binding protein that facilitates transcriptional termination and reinitiation by RNA polymerase III. Native La protein fractionates into transcriptionally active and inactive forms that are unphosphorylated and phosphorylated at serine 366, respectively, as determined by enzymatic and mass spectrometric analyses. Serine 366 comprises a casein kinase II phosphorylation site that resides within a conserved region in the La proteins from several species. RNA synthesis from isolated transcription complexes is inhibited by casein kinase II-mediated phosphorylation of La serine 366 and is reversible by dephosphorylation. This work demonstrates a novel mechanism of transcriptional control at the level of recycling of stable transcription complexes.


Nature Structural & Molecular Biology | 2006

Separate RNA-binding surfaces on the multifunctional La protein mediate distinguishable activities in tRNA maturation

Ying Huang; Mark A. Bayfield; Robert V. Intine; Richard J. Maraia

By sequence-specific binding to 3′ UUU-OH, the La protein shields precursor (pre)-RNAs from 3′ end digestion and is required to protect defective pre–transfer RNAs from decay. Although La is comprised of a La motif and an RNA-recognition motif (RRM), a recent structure indicates that the RRM β-sheet surface is not involved in UUU-OH recognition, raising questions as to its function. Progressively defective suppressor tRNAs in Schizosaccharomyces pombe reveal differential sensitivities to La and Rrp6p, a 3′ exonuclease component of pre-tRNA decay. 3′ end protection is compromised by mutations to the La motif but not the RRM surface. The most defective pre-tRNAs require a second activity of La, in addition to 3′ protection, that requires an intact RRM surface. The two activities of La in tRNA maturation map to its two conserved RNA-binding surfaces and suggest a modular model that has implications for its other ligands.


Wiley Interdisciplinary Reviews - Rna | 2011

3′ processing of eukaryotic precursor tRNAs

Richard J. Maraia; Tek N. Lamichhane

Biogenesis of eukaryotic tRNAs requires transcription by RNA polymerase III and subsequent processing. 5′ processing of precursor tRNA occurs by a single mechanism, cleavage by RNase P, and usually occurs before 3′ processing although some conditions allow observation of the 3′‐first pathway. 3′ processing is relatively complex and is the focus of this review. Precursor RNA 3′ end formation begins with pol III termination generating a variable length 3′ oligo(U) tract that represents an underappreciated and previously unreviewed determinant of processing. Evidence that the pol III‐intrinsic 3′ exonuclease activity mediated by Rpc11p affects 3′ oligo(U) length is reviewed. In addition to multiple 3′ nucleases, precursor tRNA (pre‐tRNA) processing involves La and Lsm, distinct oligo(U)‐binding proteins with proposed chaperone activities. 3′ processing is performed by the endonuclease RNase Z or the exonuclease Rex1p (possibly others) along alternate pathways conditional on La. We review a Schizosaccharomyces pombe tRNA reporter system that has been used to distinguish two chaperone activities of La protein to its two conserved RNA binding motifs. Pre‐tRNAs with structural impairments are degraded by a nuclear surveillance system that mediates polyadenylation by the TRAMP complex followed by 3′ digestion by the nuclear exosome which appears to compete with 3′ processing. We also try to reconcile limited data on pre‐tRNA processing and Lsm proteins which largely affect precursors but not mature tRNAs. A pathway is proposed in which 3′ oligo(U) length is a primary determinant of La binding with subsequent steps distinguished by 3′ endo versus exo nucleases, chaperone activities, and nuclear surveillance. WIREs RNA 2011 2 362–375 DOI: 10.1002/wrna.64


Molecular and Cellular Biology | 2004

CK2 Is Responsible for Phosphorylation of Human La Protein Serine-366 and Can Modulate rpL37 5′-Terminal Oligopyrimidine mRNA Metabolism

Elena Schwartz; Robert V. Intine; Richard J. Maraia

ABSTRACT La protein binds precursors to 5S rRNA, tRNAs, and other transcripts that contain 3′ UUU-OH and also promotes their maturation in the nucleus. Separate from this function, human La has been shown to positively modulate the translation of mRNAs that contain complex 5′ regulatory motifs that direct internal initiation of translation. Nonphosphorylated La (npLa) inhibits pre-tRNA processing, while phosphorylation of human La serine-366 (S366) promotes pre-tRNA processing. npLa was found specifically associated with a class of mRNAs that have unusually short 5′ untranslated regions comprised of terminal oligopyrimidine (5′TOP) tracts and that encode ribosomal proteins and translation elongation factors. Although La S366 represents a CK2 phosphorylation site, there was no evidence that CK2 phosphorylates it in vivo. We used the CK2-specific inhibitor, 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), and antisense-mediated knockdown to demonstrate that CK2 is responsible for La S366 phosphorylation in vivo. Hypophosphorylation was not associated with significant change in total La levels or proteolytic cleavage. Quantitative reverse transcription-PCR revealed increased association of the 5′TOP-mRNA encoding ribosomal protein L37 (rpL37) with La after TBB treatment. Transfection revealed more rpL37 mRNA associated with nonphosphorylatable La A366 than with La S366, concomitant with La A366-specific shift of a fraction of L37 mRNA off polysomes. The data indicate that CK2 phosphorylates La S366 in vivo, that this limits 5′TOP mRNA binding, and that increasing npLa leads to greater association with potentially negative effects on TOP mRNA translation. Consistent with data that indicate that phosphorylation reverses negative effects of npLa on tRNA production, the present data suggest that CK2 phosphorylation of La can affect production of the translational machinery.


PLOS Genetics | 2014

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

John W. Yarham; Tek N. Lamichhane; Angela Pyle; Sandy Mattijssen; Enrico Baruffini; Francesco Bruni; Claudia Donnini; Alex Vassilev; Langping He; Emma L. Blakely; Helen Griffin; Mauro Santibanez-Koref; Laurence A. Bindoff; Ileana Ferrero; Patrick F. Chinnery; Robert McFarland; Richard J. Maraia; Robert W. Taylor

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.


Molecular and Cellular Biology | 2005

Mutations in the RNA Polymerase III Subunit Rpc11p That Decrease RNA 3 Cleavage Activity Increase 3-Terminal Oligo(U) Length and La-Dependent tRNA Processing

Ying Huang; Robert V. Intine; Amy Mozlin; Samuel A. Hasson; Richard J. Maraia

ABSTRACT Termination by RNA polymerase III (Pol III) produces RNAs whose 3′ oligo(U) termini are bound by La protein, a chaperone that protects RNAs from 3′ exonucleases and promotes their maturation. Multiple reports indicate that yeasts use La-dependent and -independent pathways for tRNA maturation, with defective pre-tRNAs being most sensitive to decay and most dependent on La for maturation and function. The Rpc11p subunit of Pol III shows homology with the zinc ribbon of TFIIS and is known to mediate RNA 3′ cleavage and to be important for termination. We used a La-dependent opal suppressor, tRNASerUGAM, which suppresses ade6-704 and the accumulation of red pigment, to screen Schizosaccaromyces pombe for rpc11 mutants that increase tRNA-mediated suppression. Analyses of two zinc ribbon mutants indicate that they are deficient in Pol III RNA 3′ cleavage activity and produce pre-tRNASerUGAM transcripts with elongated 3′-oligo(U) tracts that are better substrates for La. A substantial fraction of pre-tRNASerUGAM contains too few 3′ Us for efficient La binding and appears to decay in wild-type cells but has elongated oligo(U) tracts and matures along the La-dependent pathway in the mutants. The data indicate that Rpc11p limits RNA 3′-U length and that this significantly restricts pre-tRNAs to a La-independent pathway of maturation in fission yeast.


Journal of Virology | 2008

Cellular La Protein Shields Nonsegmented Negative-Strand RNA Viral Leader RNA from RIG-I and Enhances Virus Growth by Diverse Mechanisms

Vira Bitko; Alla Musiyenko; Mark A. Bayfield; Richard J. Maraia; Sailen Barik

ABSTRACT The La antigen (SS-B) associates with a wide variety of cellular and viral RNAs to affect gene expression in multiple systems. We show that La is the major cellular protein found to be associated with the abundant 44-nucleotide viral leader RNA (leRNA) early after infection with respiratory syncytial virus (RSV), a nonsegmented negative-strand RNA virus. Consistent with this, La redistributes from the nucleus to the cytoplasm in RSV-infected cells. Upon RNA interference knockdown of La, leRNA is redirected to associate with the RNA-binding protein RIG-I, a known activator of interferon (IFN) gene expression, and this is accompanied by the early induction of IFN mRNA. These results suggest that La shields leRNA from RIG-I, abrogating the early viral activation of type I IFN. We mapped the leRNA binding function to RNA recognition motif 1 of La and showed that while wild-type La greatly enhanced RSV growth, a La mutant defective in RSV leRNA binding also did not support RSV growth. Comparative studies of RSV and Sendai virus and the use of IFN-negative Vero cells indicated that La supports the growth of nonsegmented negative-strand RNA viruses by both IFN suppression and a potentially novel IFN-independent mechanism.

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James R. Iben

National Institutes of Health

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Robert V. Intine

National Institutes of Health

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Ying Huang

State University of New York Upstate Medical University

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Keshab Rijal

National Institutes of Health

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Tek N. Lamichhane

National Institutes of Health

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Amanda K. Crawford

National Institutes of Health

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Amy L. Sakulich

National Institutes of Health

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