Structural basis of 3'-end poly(A) RNA recognition by LARP1.

La-related proteins (LARPs) comprise a family of RNA-binding proteins involved in a wide range of posttranscriptional regulatory activities. LARPs share a unique tandem of two RNA-binding domains, La motif (LaM) and RNA recognition motif (RRM), together referred to as a La-module, but vary in member-specific regions. Prior structural studies of La-modules reveal they are pliable platforms for RNA recognition in diverse contexts.

The isolated La-module of LARP1 mediates 3' poly(A) protection and mRNA stabilization, dependent on its intrinsic PAM2 binding to PABPC1.

The protein domain arrangement known as the La-module, comprised of a La motif (LaM) followed by a linker and RNA recognition motif (RRM), is found in seven La-related proteins: LARP1, LARP1B, LARP3 (La protein), LARP4, LARP4B, LARP6, and LARP7 in humans. Several LARPs have been characterized for their distinct activity in a specific aspect of RNA metabolism. The La-modules vary among the LARPs in linker length and RRM subtype.

Transcriptome-wide stability analysis uncovers LARP4-mediated NFκB1 mRNA stabilization during T cell activation.

T cell activation is a well-established model for studying cellular responses to exogenous stimulation. Motivated by our previous finding that intron retention (IR) could lead to transcript instability, in this study, we performed BruChase-Seq to experimentally monitor the expression dynamics of nascent transcripts in resting and activated CD4+ T cells. Computational modeling was then applied to quantify the stability of spliced and intron-retained transcripts on a genome-wide scale.

mRNA codon-tRNA match contributes to LARP4 activity for ribosomal protein mRNA poly(A) tail length protection.

Messenger RNA function is controlled by the 3' poly(A) tail (PAT) and poly(A)-binding protein (PABP). La-related protein-4 (LARP4) binds poly(A) and PABP. mRNA contains a translation-dependent, coding region determinant (CRD) of instability that limits its expression.

La Deletion from Mouse Brain Alters Pre-tRNA Metabolism and Accumulation of Pre-5.8S rRNA, with Neuron Death and Reactive Astrocytosis.

Human La antigen (Sjögren's syndrome antigen B [SSB]) is an abundant multifunctional RNA-binding protein. In the nucleoplasm, La binds to and protects from 3' exonucleases, the ends of precursor tRNAs, and other transcripts synthesized by RNA polymerase III and facilitates their maturation, while a nucleolar isoform has been implicated in rRNA biogenesis by multiple independent lines of evidence. We showed previously that conditional La knockout (La cKO) from mouse cortex neurons results in defective tRNA processing, although the pathway(s) involved in neuronal loss thereafter was unknown.

Targeted deletion of the gene encoding the La autoantigen (Sjögren's syndrome antigen B) in B cells or the frontal brain causes extensive tissue loss.

La antigen (Sjögren's syndrome antigen B) is a phosphoprotein associated with nascent precursor tRNAs and other RNAs, and it is targeted by autoantibodies in patients with Sjögren's syndrome, systemic lupus erythematosus, and neonatal lupus. Increased levels of La are associated with leukemias and other cancers, and various viruses usurp La to promote their replication. Yeast cells (Saccharomyces cerevisiae and Schizosaccharomyces pombe) genetically depleted of La grow and proliferate, whereas deletion from mice causes early embryonic lethality, raising the question of whether La is required by mammalian cells generally or only to surpass a developmental stage.

La-related protein 4 binds poly(A), interacts with the poly(A)-binding protein MLLE domain via a variant PAM2w motif, and can promote mRNA stability.

The conserved RNA binding protein La recognizes UUU-3'OH on its small nuclear RNA ligands and stabilizes them against 3'-end-mediated decay. We report that newly described La-related protein 4 (LARP4) is a factor that can bind poly(A) RNA and interact with poly(A) binding protein (PABP). Yeast two-hybrid analysis and reciprocal immunoprecipitations (IPs) from HeLa cells revealed that LARP4 interacts with RACK1, a 40S ribosome- and mRNA-associated protein.

Specific recognition of RNA/DNA hybrid and enhancement of human RNase H1 activity by HBD.

Human RNase H1 contains an N-terminal domain known as dsRHbd for binding both dsRNA and RNA/DNA hybrid. We find that dsRHbd binds preferentially to RNA/DNA hybrids by over 25-fold and rename it as hybrid binding domain (HBD). The crystal structure of HBD complexed with a 12 bp RNA/DNA hybrid reveals that the RNA strand is recognized by a protein loop, which forms hydrogen bonds with the 2'-OH groups.

Structure of human RNase H1 complexed with an RNA/DNA hybrid: insight into HIV reverse transcription.

We report here crystal structures of human RNase H1 complexed with an RNA/DNA substrate. Unlike B. halodurans RNase H1, human RNase H1 has a basic protrusion, which forms a DNA-binding channel and together with the conserved phosphate-binding pocket confers specificity for the B form and 2'-deoxy DNA.

Crystal structures of RNase H bound to an RNA/DNA hybrid: substrate specificity and metal-dependent catalysis.

RNase H belongs to a nucleotidyl-transferase superfamily, which includes transposase, retroviral integrase, Holliday junction resolvase, and RISC nuclease Argonaute. We report the crystal structures of RNase H complexed with an RNA/DNA hybrid and a mechanism for substrate recognition and two-metal-ion-dependent catalysis. RNase H specifically recognizes the A form RNA strand and the B form DNA strand.

Eukaryotic RNases H1 act processively by interactions through the duplex RNA-binding domain.

Ribonucleases H have mostly been implicated in eliminating short RNA primers used for initiation of lagging strand DNA synthesis. Escherichia coli RNase HI cleaves these RNA-DNA hybrids in a distributive manner. We report here that eukaryotic RNases H1 have evolved to be processive enzymes by attaching a duplex RNA-binding domain to the RNase H region.

Selective inhibition of HIV-1 reverse transcriptase-associated ribonuclease H activity by hydroxylated tropolones.

High-throughput screening of a National Cancer Institute library of pure natural products identified the hydroxylated tropolone derivatives beta-thujaplicinol (2,7-dihydroxy-4-1(methylethyl)-2,4,6-cycloheptatrien-1-one) and manicol (1,2,3,4-tetrahydro-5-7-dihydroxy-9-methyl-2-(1-methylethenyl)-6H-benzocyclohepten-6-one) as potent and selective inhibitors of the ribonuclease H (RNase H) activity of human immunodeficiency virus-type 1 reverse transcriptase (HIV-1 RT). beta-Thujaplicinol inhibited HIV-1 RNase H in vitro with an IC50 of 0.2 microM, while the IC50 for Escherichia coli and human RNases H was 50 microM and 5.

A capillary electrophoretic assay for ribonuclease H activity.

A capillary electrophoretic assay was developed to measure the ribonuclease (RNase) H activity of human immunodeficiency virus (HIV) type 1 reverse transcriptase. Cleavage of a fluorescein-labeled RNA-DNA heteroduplex was monitored by capillary electrophoresis. This new assay was used as a secondary assay to confirm hits from a high-throughput screening program.

Selective inhibitory DNA aptamers of the human RNase H1.

Human RNase H1 binds double-stranded RNA via its N-terminal domain and RNA-DNA hybrid via its C-terminal RNase H domain, the latter being closely related to Escherichia coli RNase HI. Using SELEX, we have generated a set of DNA sequences that can bind efficiently (K(d) values ranging from 10 to 80 nM) to the human RNase H1. None of them could fold into a simple perfect double-stranded DNA hairpin confirming that double-stranded DNA does not constitute a trivial ligand for the enzyme.

[Sensitized photomodification of DNA with binary systems of oligonucleotide conjugates. V. Effect of the structure of the target DNA. Quantitative photomodification].

A sensitized photomodification of several single-stranded target DNAs by binary systems of oligonucleotide conjugates complementary to the adjacent regions of DNA was performed. One of the conjugates contained a sensitizer (pyrene, anthracene, or 1,2-benzanthracene), and another conjugate contained a photoreagent 4-azidotetrafluorobenzalhydrazone. The sensitized photomodification is initiated by irradiation at 365-580 nm due to effective energy transfer from the excited sensitizer to the photoreagent in a complementary complex of the binary system with the target DNA where the sensitizer and photoreagent are brought sterically together.

[Sensitized photomodification of DNA with binary systems of oligonucleotide conjugates. IV. Photoinduced electron transfer].

The photomodification of single-stranded DNA sensitized to visible light (450-580 nm) by a binary system of oligonucleotide conjugates complementary to adjacent DNA sequences was studied. One oligonucleotide carries a residue of the photoreagent p-azidotetrafluorobenzaldehyde hydrazone at its 3'-terminal phosphate, and the other has a residue of the sensitizer, perylene or 1,2-benzanthracene, at the 5'-terminal phosphate. The rate of photomodification sensitized by the perylene derivative is 300,000-fold higher than the rate of photomodification in the absence of the sensitizer.

[Sensitized photomodification of DNA with binary systems of oligonucleotide conjugates. III. Double-quantum sensitization].

Site-specific modification of single-stranded DNA by oligonucleotide derivatives of p-azido-O-(4-aminobutyl)tetrafluorobenzaldoxime sensitized by an oligonucleotide derivative of pyrenylethylamine was studied. Upon irradiation with the long-wave UV light (365-390 nm) of a DNA target-oligonucleotide reagent complementary complex, a considerable increase in the rate of sensitized photomodification at the G11 residue of the target relative to the direct photomodification was observed owing to the singlet-single energy transfer from the sensitizer onto the photoreagent. Upon simultaneous irradiation of the complex with UV and visible light in the region of the triplet-triplet absorption of pyrene (360-580 nm), an additional increase in the modification rate and a change in its site-direction (from the G11 to T13 residue) occurred through the two-photon triplet-triplet sensitization.

Sensitized photomodification of single-stranded DNA by a binary system of oligonucleotide conjugates.

A photoactivatable binary system of oligonucleotide conjugates that form reactive species when assembling on a target nucleotide sequence has been developed. The binary system consists of two oligonucleotides. One contains a photosensitizing group, and the second contains a photoreactive group.

[Sensitized photomodification of DNA with binary systems. II. Spectral photosensitivity. One- and two quantum sensitization].

The efficiency of the photomodification of target single-stranded DNA with a decanucleotide derivative of p-azidotetrafluorobenzamide (direct photomodification) and with its complexes with decanucleotide derivatives of pyrene complementary to the adjacent segment of the target (sensitized photomodification) was studied as a function of the wavelength of long-wave UV light. The sensitized photomodification occurs mainly by singlet-singlet energy transfer from pyrene to azide in their complementary complex, which allows a significant increase in the rate and level of photomodification. When irradiation occurred simultaneously in the UV and visible regions (365-580 nm), two-photon triplet-triplet sensitization was revealed for the first time, which leads to a still greater acceleration of the target modification and a change of its site-direction from the G11 to T13 residue.

[Sensitized photomodification by binary systems. I. Synthesis of oligonucleotide reagents, and the effect of their structure on the efficacy of target modification].

A highly effective sensitized photomodification of the target DNA by a binary system of oligonucleotide reagents complementary to adjacent regions of the target was accomplished. One of the oligonucleotides carries a photoregent p-azidotetrafluorobenzamide, and the other carries a pyrene sensitizer. Synthesis of the oligonucleotide derivatives was described.

[Complementary addressed modification of plasmid DNA].

The possibility to accomplish the sequence-specific chemical modification of superhelical DNA with reactive oligonucleotide derivatives was demonstrated. Plasmids containing fragments of the immunoglobulin gene were modified with alkylating derivatives of oligonucleotides complementary to a nucleotide sequence in the immunoglobulin gene. In contrast to the relaxed plasmid DNAs, superhelical DNAs (sigma = -0.