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PubMedDate not supplied

A new strategy for site-specific protein modification: analysis of a Tat peptide-TAR RNA interaction.

Site-specific modification of proteins and peptides with reporter molecules provides a powerful research tool in chemistry and biology. We report the synthesis and application of a tyrosine analogue, N-alpha-Fmoc-3-acetyl-L-tyrosine, for selective modification of proteins. As a model system, we synthesized the human immunodeficiency virus type 1 (HIV-1) Tat peptide (amino acids 47-56) containing the arginine rich RNA-binding region and replaced the Tyr-47 with 3-acetyl-tyrosine. The acetyl-Tyr-Tat peptide was subsequently labeled with a fluorescein derivative to study RNA-protein interactions by fluorescence energy transfer experiments. Our results showed that the Tat peptide binds to the rhodamine labeled TAR RNA with a dissociation constant (KD) of 1.0 +/- 0.5 nM. This strategy of selective protein modification offers a versatile new procedure for labeling peptides of biological interest at a desired site when several nucleophilic side chains of lysine and cysteine are present. These methods would provide tools for postsynthetic peptide modification and introducing biophysical probes for structural and functional analysis of proteins.

Contrast Media↗
PubMed1998

Modification of U6 spliceosomal RNA is guided by other small RNAs.

The vertebrate spliceosomal snRNAs are highly modified by pseudouridylation and 2'-O-methylation. We have identified novel conserved small RNAs that can direct addition of two methyl groups in U6 snRNA, at A47 and C77. These guide RNAs, mgU6-47 (methylation guide for U6 snRNA residue 47) and mgU6-77 contain boxes C, C', D, and D' and associate with fibrillarin. Each RNA can form a duplex with U6 snRNA positioning A47 and C77 for 2'-O-methylation. The antisense element of mgU6-77 can also position C2970 of 28S rRNA for 2'-O-methylation. Depletion of mgU6-77 from Xenopus oocytes prevents 2'-O-methylation of both C77 in U6 and C2970 in 28S; methylation can be restored by injecting in vitro transcribed mgU6-77. Thus, mgU6-77 appears to function in the 2'-O-methylation of two distinct classes of cellular RNA, snRNA, and rRNA.

Animals↗
PubMed2024

m6A modification of mutant huntingtin RNA promotes the biogenesis of pathogenic huntingtin transcripts.

In Huntington's disease (HD), aberrant processing of huntingtin (HTT) mRNA produces HTT1a transcripts that encode the pathogenic HTT exon 1 protein. The mechanisms behind HTT1a production are not fully understood. Considering the role of m6A in RNA processing and splicing, we investigated its involvement in HTT1a generation. Here, we show that m6A methylation is increased before the cryptic poly(A) sites (IpA1 and IpA2) within the huntingtin RNA in the striatum of Hdh+/Q111 mice and human HD samples. We further assessed m6A's role in mutant Htt mRNA processing by pharmacological inhibition and knockdown of METTL3, as well as targeted demethylation of Htt intron 1 using a dCas13-ALKBH5 system in HD mouse cells. Our data reveal that Htt1a transcript levels are regulated by both METTL3 and the methylation status of Htt intron 1. They also show that m6A methylation in intron 1 depends on expanded CAG repeats. Our findings highlight a potential role for m6A in aberrant splicing of Htt mRNA.

Huntingtin Protein↗
PubMed1997

Polyphosphate kinase is a component of the Escherichia coli RNA degradosome.

The Escherichia coli degradosome is a multienzyme complex with four major protein components: the endoribonuclease RNase E, the exoribonuclease PNPase, the RNA helicase RhlB and enolase. The first three of these proteins are known to have important functions in mRNA processing and degradation. In this work, we identify an additional component of the degradosome, polyphosphate kinase (PPK), which catalyses the reversible polymerization of the gamma-phosphate of ATP into polyphosphate (poly(P)). An E. coli strain deleted for the ppk gene showed increased stability of the ompA mRNA. Purified His-tagged PPK was shown to bind RNA, and RNA binding was prevented by hydrolysable ATP. Chemical modification of RNA by PPK, for example the addition or removal of 3' or 5' terminal phosphates, could not be detected. However, polyphosphate was found to inhibit RNA degradation by the degradosome in vitro. This inhibition was overcome by the addition of ADP, required for the degradation of polyphosphate and for the regeneration of ATP by PPK in the degradosome. Thus, PPK in the degradosome appears to maintain an appropriate microenvironment, removing inhibitory polyphosphate and NDPs and regenerating ATP.

Adenosine Triphosphate↗
PubMed1998

Site-specific modification of 4.5S RNA apical domain by complementary oligodeoxynucleotides carrying an alkylating group.

Site-specific alkylation of RNA by reactive oligodeoxynucleotides provides structural information and represents the first step towards the design of RNA derivatives to be used for functional studies. Specific alkylation of 4.5S RNA at G53, the first base of the apical tetraloop, was achieved by incubation with oligodeoxynucleotide ON2, complementary to nucleotides 38-53, which carries a p-(N-2-chloroethyl-N-methylamino)benzylamidophosphate group at the 5' end. Alkylation efficiency was increased by a factor of 6, without alteration of specificity, in the presence of a helper oligodeoxynucleotide, ON1, complementary to nucleotides 58-71 of the opposite strand of the RNA helix. A second reactive oligodeoxynucleotide, ON1-3'-R, was obtained by attaching the alkylating group to the 3' end of ON1. ON1-3'-R was able to modify G58. In the presence of ON2 as a helper oligodeoxynucleotide, the specificity of ON1-3'-R changes and efficient alkylation of nucleotides G54, A56 and G57 of the apical region of 4.5S RNA was observed.

Alkylating Agents↗
PubMed1985

Efficient modification of E. coli RNA polymerase in vitro by the N gene transcription antitermination protein of bacteriophage lambda.

The N gene protein of bacteriophage lambda prevents termination of transcription by E. coli RNA polymerase. We describe here the conditions of a cell-free reaction system in which pure N stimulates net transcription up to tenfold and therefore nearly stoichiometrically modifies transcribing RNA polymerase molecules. The reaction contains micrococcal nuclease-treated S100 extract derived from E. coli and a plasmid template DNA containing the lambda early promoter PL, the N utilization site nutL, and the Rho-dependent terminator tL1. Stimulation by N in this system is specific and biologically relevant since it is absent with vector pBR322 DNA and with extracts derived from E. coli strains bearing the nusA1 and nusE71 mutations known to block N function in vivo. We use the system to provide further evidence that ribosomes are not necessary for N function and to demonstrate the direct involvement in N function of the NusA protein of E. coli.

Ammonia↗