Direct comparison of nick-joining activity of the nucleic acid ligases from bacteriophage T4

The genome of bacteriophage T4 encodes three polynucleotide ligases, which seal the backbone of nucleic acids during infection of host bacteria. The T4Dnl (T4 DNA ligase) and two RNA ligases [T4Rnl1 (T4 RNA ligase 1) and T4Rnl2] join a diverse array of substrates, including nicks that are present in double-stranded nucleic acids, albeit with different efficiencies. To unravel the biochemical and functional relationship between these proteins, a systematic analysis of their substrate specificity was performed using recombinant proteins. The ability of each protein to ligate 20 bp double-stranded oligonucleotides containing a single-strand break was determined. Between 4 and 37 degrees C, all proteins ligated substrates containing various combinations of DNA and RNA. The RNA ligases ligated a more diverse set of substrates than T4Dnl and, generally, T4Rnl1 had 50-1000-fold lower activity than T4Rnl2. In assays using identical conditions, optimal ligation of all substrates was at pH 8 for T4Dnl and T4Rnl1 and pH 7 for T4Rnl2, demonstrating that the protein dictates the pH optimum for ligation. All proteins ligated a substrate containing DNA as the unbroken strand, with the nucleotides at the nick of the broken strand being RNA at the 3'-hydroxy group and DNA at the 5'-phosphate. Since this RNA-DNA hybrid was joined at a similar maximal rate by T4Dnl and T4Rnl2 at 37 degrees C, we consider the possibility that this could be an unexpected physiological substrate used during some pathways of 'DNA repair'.     

Dumbbell-PCR: a method to quantify specific small RNA variants with a single nucleotide resolution at terminal sequences

Recent advances in next-generation sequencing technologies have revealed that cellular functional RNAs are not always expressed as single entities with fixed terminal sequences but as multiple isoforms bearing complex heterogeneity in both length and terminal sequences, such as isomiRs, the isoforms of microRNAs. Unraveling the biogenesis and biological significance of heterogenetic RNA expression requires distinctive analysis of each RNA variant. Here, we report the development of dumbbell PCR (Db-PCR), an efficient and convenient method to distinctively quantify a specific individual small RNA variant. In Db-PCR, 5′- and 3′-stem–loop adapters are specifically hybridized and ligated to the 5′- and 3′-ends of target RNAs, respectively, by T4 RNA ligase 2 (Rnl2). The resultant ligation products with ‘dumbbell-like’ structures are subsequently quantified by TaqMan RT-PCR. We confirmed that high specificity of Rnl2 ligation and TaqMan RT-PCR toward target RNAs assured both 5′- and 3′-terminal sequences of target RNAs with single nucleotide resolution so that Db-PCR specifically detected target RNAs but not their corresponding terminal variants. Db-PCR had broad applicability for the quantification of various small RNAs in different cell types, and the results were consistent with those from other quantification method. Therefore, Db-PCR provides a much-needed simple method for analyzing RNA terminal heterogeneity.     

Tailoring the switch from IRES-dependent to 5′-end-dependent translation with the RNase P ribozyme

Translation initiation driven by internal ribosome entry site (IRES) elements is dependent on the structural organization of the IRES region. We have previously shown that a structural motif within the foot-and-mouth-disease virus IRES is recognized in vitro as substrate for the Synechocystis sp. RNase P ribozyme. Here we show that this structure-dependent endonuclease recognizes the IRES element in cultured cells, leading to inhibition of translation. Inhibition of IRES activity was dependent on the expression of the active ribozyme RNA subunit. Moreover, expression of the antisense sequence of the ribozyme did not inhibit IRES activity, demonstrating that stable RNA structures located upstream of the IRES element do not interfere with internal initiation. RNAs carrying defective IRES mutants that were substrates of the ribozyme in vivo revealed an increased translation of the reporter in response to the expression of the active ribozyme. In support of RNA cleavage, subsequent analysis of the translation initiation manner indicated a switch from IRES-dependent to 5′-end-dependent translation of RNase P target RNAs. We conclude that the IRES element is inactivated by expression in cis of RNase P in the cytoplasm of cultured cells, providing a promising antiviral tool to combat picornavirus infections. Furthermore, our results reinforce the essential role of the structural motif that serves as RNase P recognition motif for IRES activity.     

Intracellular cleavage and ligation of hepatitis delta virus genomic RNA: regulation of ribozyme activity by cis-acting sequences and host factors.

During replication, a ribozyme within the genomic RNA of hepatitis delta virus cleaves multimeric precursors to release a unit-length linear intermediate. Intramolecular ligation of this intermediate produces the circular genomic RNA. Although one copy of the ribozyme is reconstituted by such ligation, it does not subsequently cleave and destroy the circular conformation. We have identified cis-acting attenuator sequences that prevent self-cleavage of the circular product by base pairing with and inactivating the ribozyme. Furthermore, we have shown that during the initial processing of the multimeric precursor RNA, host-specific factors activate the ribozyme by preventing its association with the attenuator sequences. Thus, we demonstrate a novel switching mechanism that regulates ribozyme activity inside the cell.     

Preferential translation of internal ribosome entry site-containing mRNAs during the mitotic cycle in mammalian cells

A cell synchronization protocol was established in which global and individual mRNA translational efficiencies could be examined. While global translational efficiency was reduced in mitotic cells, approximately 3% of mRNAs remained predominantly associated with large polysomes during mitosis, as determined by cDNA microarray analyses. The 5'-non-coding regions of six mRNAs were shown to contain internal ribosome entry sites (IRES). However, not all known mRNAs that contain IRES elements were actively translated during mitosis, arguing that specific IRES sequences are differentially regulated during mitosis.     

Observation of internal cleavage and ligation reactions of a ribozyme

We have used single-molecule spectroscopy to untangle conformational dynamics and internal chemistry in the hairpin ribozyme. The active site of the ribozyme is stably formed by docking two internal loops, but upon cleavage undocking is accelerated by two orders of magnitude. The markedly different kinetic properties allow us to differentiate cleaved and ligated forms, and thereby observe multiple cycles of internal cleavage and ligation of a ribozyme in a uniquely direct way. The position of the internal equilibrium is biased toward ligation, but the cleaved ribozyme undergoes several undocking events before ligation, during which products may dissociate. Formation of the stably docked active site, rapid undocking after cleavage, and a strong bias toward ligation should combine to generate a stable circular template for the synthesis of the viral (+) strand and thus ensure a productive replication cycle.     

细胞内部核糖体进入位点研究进展

细胞内部核糖体进入位点(IRES)mRNA5’端非编码区的一段特殊的序列,它允许核糖体不从mRNA的5’到3’端阅读而直接在此序列处结合mRNA并起始翻译。本综述了IRES的发现、IRES的识别及细胞IRES的特征、作用机理、生物学意义及其生物学应用等方面的研究进展。     

Identification of the most accessible sites to ribozymes on the hepatitis C virus internal ribosome entry site

The hepatitis C virus (HCV) is a major causative agent of chronic hepatitis and hepatocellular carcinoma. The development of alternative antiviral therapies is warranted because current treatments for the HCV infection affect only a limited number of patients and lead to significant toxicities. The HCV genome is exclusively present in the RNA form; therefore, ribozyme strategies to target certain HCV sequences have been proposed as anti-HCV treatments. In this study, we determined which regions of the internal ribosome entry site (IRES) of HCV are accessible to ribozymes by employing an RNA mapping strategy that is based on a trans-splicing ribozyme library. We then discovered that the loop regions of the domain IIIb of HCV IRES appeared to be particularly accessible. Moreover, to verify if the target sites that were predicted to be accessible are truly the most accessible, we assessed the ribozyme activities by comparing not only the trans-splicing activities in vitro but also the trans-cleavage activities in cells of several ribozymes that targeted different sites. The ribozyme that could target the most accessible site identified by mapping studies was then the most active with high fidelity in cells as well as in vitro. These results demonstrate that the RNA mapping strategy represents an effective method to determine the accessible regions of target RNAs and have important implications for the development of various antiviral therapies which are based on RNA such as ribozyme, antisense, or siRNA.     

The C-terminal domain of T4 RNA ligase 1 confers specificity for tRNA repair

T4 RNA ligase 1 (Rnl1) is a tRNA repair enzyme that thwarts a tRNA-damaging host response to virus infection. The 374-aa Rnl1 protein consists of an N-terminal nucleotidyltransferase domain fused to a unique C-terminal domain composed of 10 alpha helices. We exploited an in vitro tRNA splicing system to demonstrate that Rnl1 has an inherent specificity for sealing tRNA with a break in the anticodon loop. The tRNA specificity is imparted by the C domain, any deletion of which caused the broken tRNA to be sealed as poorly as the linear intron in vitro and also abolished Rnl1 tRNA splicing activity in vivo. Deletion analysis demarcated Rnl1-(1-254) as a minimal catalytic domain of Rnl1, capable of all chemical steps of the nonspecific RNA ligation reaction. Alanine scanning of the N domain identified Ser103, Leu104, Lys117, and Ser118 as important for pRNA ligation in vitro and tRNA repair in vivo.     

Replication of hepatitis A viruses with chimeric 5' nontranslated regions.

The role of the 5' nontranslated region in the replication of hepatitis A virus (HAV) was studied by analyzing the translation and replication of chimeric RNAs containing the encephalomyocarditis virus (EMCV) internal ribosome entry segment (IRES) and various lengths (237, 151, or 98 nucleotides [nt]) of the 5'-terminal HAV sequence. Translation of all chimeric RNAs, truncated to encode only capsid protein sequences, occurred with equal efficiency in rabbit reticulocyte lysates and was much enhanced over that exhibited by the HAV IRES. Transfection of FRhK-4 cells with the parental HAV RNA and with chimeric RNA generated a viable virus which was stable over continuous passage; however, more than 151 nt from the 5' terminus of HAV were required to support virus replication. Single-step growth curves of the recovered viruses from the parental RNA transfection and from transfection of RNA containing the EMCV IRES downstream of the first 237 nt of HAV demonstrated replication with similar kinetics and similar yields. When FRhK-4 cells infected with recombinant vaccinia virus producing SP6 RNA polymerase to amplify HAV RNA were transfected with plasmids coding for these viral RNAs or with subclones containing only HAV capsid coding sequences downstream of the parental or chimeric 5' nontranslated region, viral capsid antigens were synthesized from the HAV IRES with an efficiency equal to or greater than that achieved with the EMCV IRES. These data suggest that the inherent translation efficiency of the HAV IRES may not be the major limiting determinant of the slow-growth phenotype of HAV.     

Translational efficiency of BVDV IRES and EMCV IRES for T7 RNA polymerase driven cytoplasmic expression in mammalian cell lines

Mammalian T7 polymerase-based cytoplasmic expression systems are common tool for molecular studies. The majority of these systems include the internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). To carry out a cap-independent translation process, this type of IRES might require the expression of an extensive array of host factors, what is a disadvantage. Other IRESes might be less dependent on the host cell factors, but their biology is characterized to a lesser degree. Here, we compare the translational efficiencies of bovine viral diarrhea virus (BVDV) IRES with that of ECMV. Both IRESes were tested in reporter vectors containing the T7 promoter, an IRES of choice and the coding sequence of the enhanced green fluorescent protein (EGFP). To provide for the expression of T7 RNA polymerase, the corresponding gene was isolated from Escherichia coli and inserted into pCDNA3.1-Hygro(+). After co-transfection of the T7 RNA polymerase encoding vector with either of the two IRES-containing reporter vectors into T7 baby hamster kidney (T7-BHK), human embryonic kidney (HEK) 293T, chinese hamster ovary (CHO) and HeLa cells, the translational efficiency of the reporter construct was studied by fluorescence microscopy and flow cytometry. In T7-BHK, HEK 293T and HeLa cells the translational efficiency of BVDV IRES was two to three times higher than that of EMCV IRES. In CHO cells, BVDV IRES and EMCV IRES were equally efficient. An analysis of the secondary structure of respective mRNAs showed that their ΔG values were–544.00 and–469.40 kcal/mol for EMCV IRES and BVDV IRES harboring molecules, respectively. As EMCV IRES-containing mRNA is more stable, it is evident that other, still unidentified factors should be held responsible for the enhanced translational efficiency of BDVD IRES. Taken together, our results indicate the potential of BVDV IRES as a replacement for EMCV IRES, which is now commonly used for T7 polymerase driven cytoplasmic expression of genes of interest or virus cDNA rescue experiments.     

Covalent ligation studies on the human telomere quadruplex

Recent X-ray crystallographic studies on the human telomere sequence d[AGGG(TTAGGG)₃] revealed a unimolecular, parallel quadruplex structure in the presence of potassium ions, while earlier NMR results in the presence of sodium ions indicated a unimolecular, antiparallel quadruplex. In an effort to identify and isolate the parallel form in solution, we have successfully ligated into circular products the single-stranded human telomere and several modified human telomere sequences in potassium-containing solutions. Using these sequences with one or two terminal phosphates, we have made chemically ligated products via creation of an additional loop. Circular products have been identified by polyacrylamide gel electrophoresis, enzymatic digestion with exonuclease VII and electrospray mass spectrometry in negative ion mode. Optimum pH for the ligation reaction of the human telomere sequence ranges from 4.5 to 6.0. Several buffers were also examined, with MES yielding the greatest ligation efficiency. Human telomere sequences with two phosphate groups, one each at the 3′ and 5′ ends, were more efficient at ligation, via pyrophosphate bond formation, than the corresponding sequences with only one phosphate group, at the 5′ end. Circular dichroism spectra showed that the ligation product was derived from an antiparallel, single-stranded guanine quadruplex rather than a parallel single-stranded guanine quadruplex structure.     

The role of mRNA 5'-noncoding and 3'-end sequences on 40S ribosomal subunit recruitment,and how RNA viruses successfully compete with cellular mRNAs to ensure their own protein synthesis

Since the elaboration of the scanning model to explain eukaryotic translation initiation, alternative hypotheses have gained support. Cap and 5' end-independent recruitment of the 40S ribosomal subunit conferred by the presence of an internal ribosome entry segment (IRES) in the 5'UTR of the mRNA is widely accepted, and has been formally and definitively proven for a picornavirus. However, the mechanism of IRES function remains essentially a black box. Using the complex viral IRESes as model systems, approaches taken to shed light on the mystery include systematic comparisons and molecular genetic analyses. The hypothesis that actively translated mRNAs are circular, rather than linear, molecules is based on rather indirect evidence. This model has invoked a revision of the image of 40S ribosomal subunit recruitment, to include recycling from the mRNA 3'- to the 5'-end in addition to true de novo 5'-end directed entry. Biochemical and genetic studies are used to define the network of interactions necessary for efficient ribosome recruitment. This has lent weight to the concept of mRNA 5'-3' cross-talk and clarified the mechanics of how this enhances translation efficiency. These refinements and revisions to the model of translation initiation form the core of this review, with current knowledge being considered from the perspective on how host-cell translation could yield to selective viral translation via the phenomenon of translational shut-off.