GUPTA LAB

Three Domains of Life
Our research interests are in the area of gene regulation in Archaea (Archaebacteria), with emphasis on RNA processing. Archaea are one of the three domains of life, the other two being Bacteria (eubacteria) and Eukarya (eukaryotes). Most Archaea grow under extreme environmental conditions. These include methanogens (methane producers), extreme halophiles (grow in 2M to nearly saturated salt concentrations), thermophiles and hyperthermophiles (grow at 65 to 105ºC), acidophiles, alkaliphiles, etc. Archaea, like Bacteria, are prokaryotic in organization, but show similarities to several components of the eukaryotic replication, transcription and translation systems.

RNA Processing
Posttranscriptional RNA processing involves additions and deletions at the ends of the transcripts, RNA splicing, RNA editing, residue modification, etc. Previously, we have sequenced a nearly complete set of tRNAs from a halophilic archaeon -- Haloferax volcanii (Halobacterium volcanii), characterized several modified nucleotides present in archaeal tRNAs, and reported the presence of introns in several archaeal tRNA genes. Our current research interests involve RNA splicing, sRNA-guided and guide RNA-independent modification of RNA residues and their roles in the biology of Archaea.

RNA Splicing in Archaea
Splicing of introns in Archaea involves an endonuclease and a ligase. We have established an in vitrosystem to study RNA splicing in halophilic Archaea. Using this system we have shown that during ligation, the phosphate at the splicing junction is derived from the precursor RNA. This is also the case for the animal type tRNA splicing ligase, which is different from the tRNA splicing ligase of yeast. We have also shown that during splicing not only the two exons ligate together but the intron-ends also join to form circular RNAs. In some cases these circular products are retained in the cell, suggesting that they may have a functional role.
Work from our laboratory (unpublished) and other laboratories has shown that ribosomal RNA processing in Archaea also involves splicing endonuclease and ligase activity.

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A model for the reactions occurring during RNA splicing in Archaea (A) Symmetric natures of the bulge-helix-bulge (BHB) containing endonuclease substrate and the two seven-base hairpin loops in the ligase products. Arrows indicate the splice sites in the substrate and the asterisks indicate the junction phosphates in the products. (B) Reactions involving specific phosphodiester linkages during RNA splicing. 1–9 and 1*–9* are residues involved in the formation of BHB in the substrates and hairpins in the products. The two phosphates (p1 and p2) and 2‘, 3', and 5' positions positions involved in the reactions are indicated. (E1 and E2) two exons; (I) intron.

Eukaryotic snoRNAs and Archaeal sRNAs
The tRNAs and rRNAs of all organisms contain many different types of nucleotide modifications. In eukaryotes most of the rRNA 2'-O-methylation and pseudouridylation are carried out by snoRNPs (small nucleolar ribonucleoproteins), the RNA components of which function as guides to select the sites of target rRNA modification. Box C/D RNAs guide 2'-O-methylations and box H/ACA RNAs guide pseudouridylations. Homologs of both box C/D and H/ACA snoRNAs and their associated proteins have been reported in Archaea. These RNAs are known as sRNAs (small nucleolar RNA like RNAs) in Archaea.

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Primary sequences and predicted secondary structure of the H. volcanii pre-tRNAs. (A) The intron of the pre-tRNATrpis a box C/D RNA. The tRNA anticodon sequence (CCA) is indicated in large letters in the pre-tRNA. The exon-intron junctions, designated by arrows, are located within the bulge-helix-bulge structure required for pre-tRNA splicing. Boxes C, D, C', and D' are enclosed and designated. Complementary guide and target sequences are designated by the thick lines (box C/D) and thin lines (C'/D'). The target nucleotides in the pre-tRNA are numbered C34 and U39 according to the standard tRNA numbering system. Complementary guide (lower case) and target (upper case) nucleotide pairs (g117:C34 and a70:U39) are indicated in black squares (C/D motif) and black circles (C'/D' motif), respectively. (B)A box H/ACA RNA that guides pseudouridylation of U2605 and U1940/1942 of 23S rRNA. The spacer region between the two stem-loop structures can potentially form a psedoknot (inset). Conserved box H and ACA are underlined. Conserved G•A pairs in the K-turn structures re boxed.

sRNA-guided 2'-O-methylation in Archaea
The intron of pre-tRNATrp of Haloferax volcanii has features of box C/D sRNA and is reported to guide 2'-O-methylation of targets in its exons. Our earlier results have shown that, in vivo, residues at positions 34 and 39 of the mature tRNATrp of H. volcanii are 2'-O-methylated. We have developed a heterologous, in vitro modification system using Methanocaldococcus jannaschii proteins and pre-tRNATrp from H. volcanii. Using this system, we have proven that 2'-O-methylations at positions 34 and 39 of the pre-tRNA occur through guide-target pairing in trans, although both guide and targets are present in cis in the pre-tRNA. The intron as part of the pre-tRNA or in free linear or circular form, produced during splicing reaction can act as guide in these in vitro reactions. Using the same system we have also shown that methylation of the two nucleotides, guided by the two sites of a single box C/D RNA occur sequentially. Box C'/D' RNP-guided U39 methylation first requires a box C/D RNP-guided methylation of C34. We also show that dynamic guide-target interactions contribute to this sequential modification. Based on these and earlier results (retention of circularized introns in vivo) we propose an in vivo scenario, where the intron splicing and RNA methylation events occur in concert. In addition, we have also constructed an H. volcanii strain that has deletion of intron in its pre-tRNATrp gene. As expected, this strain lacked 2'-O-methylations at positions 34 and 39 of its tRNATrp. Surprisingly, the strain showed no detectable phenotype, in spite of deletion within a single-copy essential gene in the genome. We are presently studying the structural features of the guide/target RNAs that help to recruit proteins for efficient catalysis. Also, we have collaborated successfully with other laboratories to create deletion strains of some of the core box C/D proteins. We are now characterizing these deletion strains.
H. volcanii elongator tRNAMet also contains a 2'-O-methylated C residue at position 34. But unlike tRNATrp, this modification is not intron-mediated, but is brought about by another box C/D guide RNA in the cell that we named sR-tMet. H. volcanii sR-tMet has certain features distinct from most of the box C/D RNAs. Its uniqueness has enabled us to use it as a model RNA for initiating several structural studies. We have recently been able to create a genomic deletion of this RNA in H. volcanii. Currently we are in the process of doing several mutational studies in vivo to identify and characterize the structural motifs/ residues important for its function.

sRNA-guided pseudouridylation in rRNAs of Archaea
In archaeal rRNAs, as in the eukaryotic rRNAs, certain pseudouridines (Ψ) are produced by RNPs containing box H/ACA RNAs. The catalytic protein in these RNPs is called Cbf5. We have been able to delete Cbf5 in H. volcanii, which causes loss of predicted Ψ in its rRNA. This protein is essential in eukaryotes and cannot be deleted. We have established an in vivo system to study the functions of several critical structural domains of Cbf5. We have also identified a box H/ACA RNA of H. volcanii that functions as guide RNA for several Y modifications of the rRNA and are studying structure/function relationship of its various motifs.

Proposed model for the trans-2'-O-methylation of H. volcanii pre-tRNATrp in vivo. Thick arrows indicate the pre-tRNATrp processing pathway proceeding from transcription of the pre-tRNA through nucleotide methylation and splicing to the production of tRNATrp and excised intron. RNP assembly is denoted by thin arrows, and nucleotide modification guided intermolecularly by the intron-encoded box C/D RNP is denoted by dashed arrows.

Pseudouridine formation in tRNAs of Archaea
Pseudouridine (ψ) is almost universally present at position 55 of tRNAs in all three domains of life. Bacterial TruB protein and its homolog Pus4 in yeast convert U at this position to ψ. This reaction is catalyzed in Archaea by the Pus10 protein, which is not a member of TruB/Pus4 family of pseudouridine synthases. Pus10 homologs are found in most Archaea and eukaryotes, but not in Bacteria and yeast. This coincides with the presence of ψ54 in most tRNAs of Archaea and a few tRNAs of higher eukaryotes and its absence in Bacteria and yeast. Instead, most tRNAs of Bacteria and eukaryotes contain ribothymidine at position 54. We have shown that archaeal Pus10 proteins can produce ψ54 in addition to its tRNAs Y55 synthase activity. Recently we also showed that a tRNA methyltransferase TrmY is responsible for converting this ψ54 into hypermodified m1ψ54. The homology of eukaryotic Pus10 with archaeal Pus10 suggests that the former may also have a tRNA ψ54 synthase activity. We initiated several mutational studies of archaeal Pus10 protein and were successful in identifying certain domains/motifs of the protein that serve as crucial determinants for its ψ54 activity. Additionally, human Pus10 is involved in TRAIL-induced apoptosis. This implies a dual functional role of Pus10 in higher eukaryotes. We are currently investigating this interesting possibility

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