Optimizing the detection of nascent transcripts by RNA fluorescence in situ hybridization

An unusual feature of the mammalian genome is the number of genes exhibiting monoallelic expression. Recently random monoallelic expression of autosomal genes has been reported for olfactory and Ly-49 NK receptor genes, as well as for Il-2, Il-4 and Pax5. RNA fluorescence in situ hybridization (FISH) has been exploited to monitor allelic expression by visualizing the number of sites of transcription in individual nuclei. However, the sensitivity of this technique is difficult to determine for a given gene. We show that by combining DNA and RNA FISH it is possible to control for the hybridization efficiency and the accessibility and visibility of fluorescent probes within the nucleus.

RNA–protein hybrid ribozymes that efficiently cleave any mRNA independently of the structure of the target RNA

Ribozyme activity in vivo depends on achieving high-level expression, intracellular stability, target colocalization, and cleavage site access. At present, target site selection is problematic because of unforeseeable secondary and tertiary RNA structures that prevent cleavage. To overcome this design obstacle, we wished to engineer a ribozyme that could access any chosen site. To create this ribozyme, the constitutive transport element (CTE), an RNA motif that has the ability to interact with intracellular RNA helicases, was attached to our ribozymes so that the helicase-bound, hybrid ribozymes would be produced in cells. This modification significantly enhanced ribozyme activity in vivo, permitting cleavage of sites previously found to be inaccessible. To confer cleavage enhancement, the CTE must retain helicase-binding activity. Binding experiments demonstrated the likely involvement of RNA helicase(s). We found that attachment of the RNA motif to our tRNA ribozymes leads to cleavage in vivo at the chosen target site regardless of the local RNA secondary or tertiary structure.

Identifying ribozyme-accessible sites using NUH triplet-targeting gapmers

Accurately identifying accessible sites in RNA is a critical prerequisite for optimising the cleavage efficiency of hammerhead ribozymes and other small nucleozymes. Here we describe a simple RNase H-based procedure to rapidly identify hammerhead ribozyme-accessible sites in gene length RNAs. Twelve semi-randomised RNA–DNA–RNA chimeric oligonucleotide probes, known as ‘gapmers’, were used to direct RNase H cleavage of transcripts with the specificity expected for hammerhead ribozymes, i.e. after NUH sites (where H is A, C or U). Cleavage sites were identified simply by the mobility of RNase H cleavage products relative to RNA markers in denaturing polyacrylamide gels. Sites were identified in transcripts encoding human interleukin-2 and platelet-derived growth factor. Thirteen minimised hammerhead ribozymes, miniribozymes (Mrz), were synthesised and in vitro cleavage efficiency (37°C, pH 7.6 and 1 mM MgCl2) at each site was analysed. Of the 13 Mrz, five were highly effective, demonstrating good initial rate constants and extents of cleavage. The speed and accuracy of this method commends its use in screening for hammerhead-accessible sites.

In vitro characterization of mutant yeast RNA polymerase II with reduced binding for elongation factor TFIIS.

We have reported previously the isolation and genetic characterization of mutations in the gene encoding the largest subunit of yeast RNA polymerase II (RNAPII), which lead to 6-azauracil (6AU)-sensitive growth. It was suggested that these mutations affect the functional interaction between RNAPII and transcription-elongation factor TFIIS because the 6AU-sensitive phenotype of the mutant strains was similar to that of a strain defective in the production of TFIIS and can be suppressed by increasing the dosage of the yeast TFIIS-encoding gene, PPR2, RNAPIIs were purified and characterized from two independent 6AU-sensitive yeast mutants and from wild-type (wt) cells. In vitro, in the absence of TFIIS, the purified wt polymerase and the two mutant polymerases showed similar specific activity in polymerization, readthrough at intrinsic transcriptional arrest sites and nascent RNA cleavage. In contrast to the wt polymerase, both mutant polymerases were not stimulated by the addition of a 3-fold molar excess of TFIIS in assays of promoter-independent transcription, readthrough or cleavage. However, stimulation of the ability of the mutant RNAPIIs to cleave nascent RNA and to read through intrinsic arrest sites was observed at TFIIS:RNAPII molar ratios greater than 600:1. Consistent with these findings, the binding affinity of the mutant polymerases for TFIIS was found to be reduced by more than 50-fold compared with that of the wt enzyme. These studies demonstrate that TFIIS has an important role in the regulation of transcription by yeast RNAPII and identify a possible binding site for TFIIS on RNAPII.

RNA replication by Q beta replicase: a working model.

Two classes of RNA ligands that bound to separate, high affinity nucleic acid binding sites on Q beta replicase were previously identified. RNA ligands to the two sites, referred to as site I and site II, were used to investigate the molecular mechanism of RNA replication employed by the four-subunit replicase. Replication inhibition by site I- and site II-specific ligands defined two subsets of replicatable RNAs. When provided with appropriate 3' ends, ligands to either site served as replication templates. UV crosslinking experiments revealed that site I is associated with the S1 subunit, site II with elongation factor Tu, and polymerization with the viral subunit of the holoenzyme. These results provide the framework for a three site model describing template recognition and product strand initiation by Q beta replicase.

Initiation of (-) strand DNA synthesis from tRNA(3Lys) on lentiviral RNAs: implications of specific HIV-1 RNA-tRNA(3Lys) interactions inhibiting primer utilization by retroviral reverse transcriptases.

Initiation of minus (-) strand DNA synthesis was examined on templates containing R, U5, and primer-binding site regions of the human immunodeficiency virus type 1 (HIV-1), feline immunodeficiency virus (FIV), and equine infectious anemia virus (EIAV) genomic RNA. DNA synthesis was initiated from (i) an oligoribonucleotide complementary to the primer-binding sites, (ii) synthetic tRNA(3Lys), and (iii) natural tRNA(3Lys), by the reverse transcriptases of HIV-1, FIV, EIAV, simian immunodeficiency virus, HIV type 2 (HIV-2), Moloney murine leukemia virus, and avian myeloblastosis virus. All enzymes used an oligonucleotide on wild-type HIV-1 RNA, whereas only a limited number initiated (-) strand DNA synthesis from either tRNA(3Lys). In contrast, all enzymes supported efficient tRNA(3Lys)-primed (-) strand DNA synthesis on the genomes of FIV and EIAV. This may be in part attributable to the observation that the U5-inverted repeat stem-loop of the EIAV and FIV genomes lacks an A-rich loop shown with HIV-1 to interact with the U-rich tRNA anticodon loop. Deletion of this loop in HIV-1 RNA, or disrupting a critical loop-loop complex by tRNA(3Lys) extended by 9 nt, restored synthesis of HIV-1 (-) strand DNA from primer tRNA(3Lys) by all enzymes. Thus, divergent evolution of lentiviruses may have resulted in different mechanisms to use the same host tRNA for initiation of reverse transcription.

Determinants of RNA polymerase alpha subunit for interaction with beta, beta', and sigma subunits: hydroxyl-radical protein footprinting.

Escherichia coli RNA polymerase (RNAP) alpha subunit serves as the initiator for RNAP assembly, which proceeds according to the pathway 2 alpha-->alpha 2-->alpha 2 beta-->alpha 2 beta beta'-->alpha 2 beta beta' sigma. In this work, we have used hydroxyl-radical protein footprinting to define determinants of alpha for interaction with beta, beta', and sigma. Our results indicate that amino acids 30-75 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interaction with beta (i.e., in alpha 2 beta, alpha 2 beta beta', and alpha 2 beta beta' sigma), and amino acids 175-210 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interaction with beta' (i.e., in alpha 2 beta beta' and alpha 2 beta beta' sigma). The protected regions are conserved in the alpha homologs of prokaryotic, eukaryotic, archaeal, and chloroplast RNAPs and contain sites of substitutions that affect RNAP assembly. We conclude that the protected regions define determinants of alpha for direct functional interaction with beta and beta'. The observed maximal magnitude of protection upon interaction with beta and the observed maximal magnitude of protection upon interaction with beta' both correspond to the expected value for complete protection of one of the two alpha protomers of RNAP (i.e., 50% protection). We propose that only one of the two alpha protomers of RNAP interacts with beta and that only one of the two alpha protomers of RNAP interacts with beta'.

Reactivation of denatured proteins by 23S ribosomal RNA: role of domain V.

Escherichia coli ribosome, its 50S subunit, or simply the 23S rRNA can reactivate denatured proteins in vitro. Here we show that protein synthesis inhibitors chloramphenicol and erythromycin, which bind to domain V of 23S rRNA of E. coli, can inhibit reactivation of denatured pig muscle lactate dehydrogenase and fungal glucose-6-phosphate dehydrogenase by 23S rRNA completely. Oligodeoxynucleotides complementary to two regions within domain V (which cover sites of chloramphenicol resistant mutations and the putative A site of the incoming aminoacyl tRNA), but not to a region outside of domain V, also can inhibit the activity. Domain V of 23S rRNA, therefore, appears to play a crucial role in reactivation of denatured proteins.

RNA binding by the Wilms tumor suppressor zinc finger proteins.

The Wilms tumor suppressor gene WT1 is implicated in the ontogeny of genito-urinary abnormalities, including Denys-Drash syndrome and Wilms tumor of the kidney. WT1 encodes Kruppel-type zinc finger proteins that can regulate the expression of several growth-related genes, apparently by binding to specific DNA sites located within 5' untranslated leader regions as well as 5' promoter sequences. Both WT1 and a closely related early growth response factor, EGR1, can bind the same DNA sequences from the mouse gene encoding insulin-like growth factor 2 (Igf-2). We report that WT1, but not EGR1, can bind specific Igf-2 exonic RNA sequences, and that the zinc fingers are required for this interaction. WT1 zinc finger 1, which is not represented in EGR1, plays a more significant role in RNA binding than zinc finger 4, which does have a counterpart in EGR1. Furthermore, the normal subnuclear localization of WT1 proteins is shown to be RNase, but not DNase, sensitive. Therefore, WT1 might, like the Kruppel-type zinc finger protein TFIIIA, regulate gene expression by both transcriptional and posttranscriptional mechanisms.

Increased accommodation of nascent RNA in a product site on RNA polymerase II during arrest.

RNA polymerases encounter specific DNA sites at which RNA chain elongation takes place in the absence of enzyme translocation in a process called discontinuous elongation. For RNA polymerase II, at least some of these sequences also provoke transcriptional arrest where renewed RNA polymerization requires elongation factor SII. Recent elongation models suggest the occupancy of a site within RNA polymerase that accommodates nascent RNA during discontinuous elongation. Here we have probed the extent of nascent RNA extruded from RNA polymerase II as it approaches, encounters, and departs an arrest site. Just upstream of an arrest site, 17-19 nucleotides of the RNA 3'-end are protected from exhaustive digestion by exogenous ribonuclease probes. As RNA is elongated to the arrest site, the enzyme does not translocate and the protected RNA becomes correspondingly larger, up to 27 nucleotides in length. After the enzyme passes the arrest site, the protected RNA is again the 18-nucleotide species typical of an elongation-competent complex. These findings identify an extended RNA product groove in arrested RNA polymerase II that is probably identical to that emptied during SII-activated RNA cleavage, a process required for the resumption of elongation. Unlike Escherichia coli RNA polymerase at a terminator, arrested RNA polymerase II does not release its RNA but can reestablish the normal elongation mode downstream of an arrest site. Discontinuous elongation probably represents a structural change that precedes, but may not be sufficient for, arrest by RNA polymerase II.

Interactions between tRNA identity nucleotides and their recognition sites in glutaminyl-tRNA synthetase determine the cognate amino acid affinity of the enzyme.

Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs both ensure accurate RNA recognition and prevent the binding of noncognate substrates. Here we show for Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) that the accuracy of tRNA recognition also determines the efficiency of cognate amino acid recognition. Steady-state kinetics revealed that interactions between tRNA identity nucleotides and their recognition sites in the enzyme modulate the amino acid affinity of GlnRS. Perturbation of any of the protein-RNA interactions through mutation of either component led to considerable changes in glutamine affinity with the most marked effects seen at the discriminator base, the 10:25 base pair, and the anticodon. Reexamination of the identity set of tRNA(Gln) in the light of these results indicates that its constituents can be differentiated based upon biochemical function and their contribution to the apparent Gibbs' free energy of tRNA binding. Interactions with the acceptor stem act as strong determinants of tRNA specificity, with the discriminator base positioning the 3' end. The 10:25 base pair and U35 are apparently the major binding sites to GlnRS, with G36 contributing both to binding and recognition. Furthermore, we show that E. coli tryptophanyl-tRNA synthetase also displays tRNA-dependent changes in tryptophan affinity when charging a noncognate tRNA. The ability of tRNA to optimize amino acid recognition reveals a novel mechanism for maintaining translational fidelity and also provides a strong basis for the coevolution of tRNAs and their cognate synthetases.

Protein-RNA interactions in the active center of transcription elongation complex.

By using a crosslinkable probe incorporated into the 3' terminus of nascent transcript, three sites were mapped in Escherichia coli RNA polymerase that are contacted by the RNA in the productive elongation complex. Two of these sites are in the beta subunit and one is in the beta' subunit. During elongation, the transcription complex occasionally undergoes an arrest whereby it can neither extend nor release the RNA transcript. It is demonstrated that in an arrested complex, the three contacts of RNA 3' terminus are lost, while a new beta' subunit contact becomes prominent. Thus, elongation arrest appears to involve the disengagement of the bulk of the active center from the 3' terminus of RNA and the transfer of the terminus into a new protein environment.

Interresidue hydrogen bonding in a peptide nucleic acid.RNA heteroduplex.

Previous molecular mechanics calculations suggest that strands of peptide nucleic acids (PNAs) and complementary oligonucleotides form antiparallel duplexes stabilized by interresidue hydrogen bonds. In the computed structures, the amide carbonyl oxygen nearest the nucleobase (O7') forms an interresidue hydrogen bond with the backbone amide proton of the following residue, (n + 1)H1'. Of the 10 published two dimensional 1H NMR structures of a hexameric PNA.RNA heteroduplex. PNA(GAACTC).r(GAGUUC), 9 exhibit two to five potential interresidue hydrogen bonds. In our minimized average structure, created from the coordinates of these 10 NMR structures, three of the five possible interresidue hydrogen bond sites within the PNA backbone display the carbonyl oxygen (O7') and the amide proton (n + 1)H1' distances and N1'-H1'-(n - 1)O7' angles optimal for hydrogen bond formation. The finding of these interresidue hydrogen bonds supports the results of our previous molecular mechanics calculations.

Binding of the sigma 70 protein to the core subunits of Escherichia coli RNA polymerase, studied by iron-EDTA protein footprinting.

We have used a nonspecific protein cleaving reagent to map the interactions between subunits of the multisubunit enzyme RNA polymerase (Escherichia coli). We developed suitable conditions for using an untethered Fe-EDTA reagent, which does not bind significantly to proteins. Comparison of the cleaved fragments of the subunits from the core enzyme (alpha 2 beta beta') and the holoenzyme (core+sigma 70) shows that absence of the sigma 70 subunit is associated with the appearance of several cleavage sites on the subunits beta (within 10 residues of sequence positions 745, 764, 795, and 812) and beta' (within 10 residues of sequence positions 581, 613, and 728). A cleavage site near beta residue 604 is present in the holoenzyme but absent in the core, demonstrating that a conformational change occurs when sigma 70 binds. No differences are observed for the alpha subunit.

Testis/brain RNA-binding protein attaches translationally repressed and transported mRNAs to microtubules.

We have previously identified a testicular phosphoprotein that binds to highly conserved sequences (Y and H elements) in the 3' untranslated regions (UTRs) of testicular mRNAs and suppresses in vitro translation of mRNA constructs that contain these sequences. This protein, testis/brain RNA-binding protein (TB-RBP) also is abundant in brain and binds to brain mRNAs whose 3' UTRs contain similar sequences. Here we show that TB-RBP binds specific mRNAs to microtubules (MTs) in vitro. When TB-RBP is added to MTs reassembled from either crude brain extracts or from purified tubulin, most of the TB-RBP binds to MTs. The association of TB-RBP with MTs requires the assembly of MTs and is diminished by colcemid, cytochalasin D, and high levels of salt. Transcripts from the 3' UTRs of three mRNAs that contain the conserved sequence elements (transcripts for protamine 2, tau protein, and myelin basic protein) are linked by TB-RBP to MTs, whereas transcripts that lack the conserved sequences do not bind TB-RBP. We conclude that TB-RBP serves as an attachment protein for the MT association of specific mRNAs. Considering its ability to arrest translation in vitro, we propose that TB-RBP functions in the storage and transportation of mRNAs to specific intracellular sites where they are translated.