T4 RNA Ligase 2 truncated active site mutants: improved tools for RNA analysis

<p>Abstract</p> <p>Background</p> <p>T4 RNA ligases 1 and 2 are useful tools for RNA analysis. Their use upstream of RNA analyses such as high-throughput RNA sequencing and microarrays has recently increased their importance. The truncated form of T4 RNA ligase 2, comprising amino acids 1-249 (T4 Rnl2tr), is an attractive tool for attachment of adapters or labels to RNA 3'-ends. Compared to T4 RNA ligase 1, T4 Rnl2tr has a decreased ability to ligate 5'-PO₄ends in single-stranded RNA ligations, and compared to the full-length T4 Rnl2, the T4 Rnl2tr has an increased activity for joining 5'-adenylated adapters to RNA 3'-ends. The combination of these properties allows adapter attachment to RNA 3'-ends with reduced circularization and concatemerization of substrate RNA.</p> <p>Results</p> <p>With the aim of further reducing unwanted side ligation products, we substituted active site residues, known to be important for adenylyltransferase steps of the ligation reaction, in the context of T4 Rnl2tr. We characterized the variant ligases for the formation of unwanted ligation side products and for activity in the strand-joining reaction.</p> <p>Conclusions</p> <p>Our data demonstrate that lysine 227 is a key residue facilitating adenylyl transfer from adenylated ligation donor substrates to the ligase. This reversal of the second step of the ligation reaction correlates with the formation of unwanted ligation products. Thus, T4 Rn2tr mutants containing the K227Q mutation are useful for reducing undesired ligation products. We furthermore report optimal conditions for the use of these improved T4 Rnl2tr variants.</p

Structural bias in T4 RNA ligase-mediated 3′-adapter ligation

T4 RNA ligases are commonly used to attach adapters to RNAs, but large differences in ligation efficiency make detection and quantitation problematic. We developed a ligation selection strategy using random RNAs in combination with high-throughput sequencing to gain insight into the differences in efficiency of ligating pre-adenylated DNA adapters to RNA 3′-ends. After analyzing biases in RNA sequence, secondary structure and RNA-adapter cofold structure, we conclude that T4 RNA ligases do not show significant primary sequence preference in RNA substrates, but are biased against structural features within RNAs and adapters. Specifically, RNAs with less than three unstructured nucleotides at the 3′-end and RNAs that are predicted to cofold with an adapter in unfavorable structures are likely to be poorly ligated. The effect of RNA-adapter cofold structures on ligation is supported by experiments where the ligation efficiency of specific miRNAs was changed by designing adapters to alter cofold structure. In addition, we show that using adapters with randomized regions results in higher ligation efficiency and reduced ligation bias. We propose that using randomized adapters may improve RNA representation in experiments that include a 3′-adapter ligation step

Group II Intron-Based Gene Targeting Reactions in Eukaryotes

Mobile group II introns insert site-specifically into DNA target sites by a mechanism termed retrohoming in which the excised intron RNA reverse splices into a DNA strand and is reverse transcribed by the intron-encoded protein. Retrohoming is mediated by a ribonucleoprotein particle that contains the intron-encoded protein and excised intron RNA, with target specificity determined largely by base pairing of the intron RNA to the DNA target sequence. This feature enabled the development of mobile group II introns into bacterial gene targeting vectors ("targetrons") with programmable target specificity. Thus far, however, efficient group II intron-based gene targeting reactions have not been demonstrated in eukaryotes.By using a plasmid-based Xenopus laevis oocyte microinjection assay, we show that group II intron RNPs can integrate efficiently into target DNAs in a eukaryotic nucleus, but the reaction is limited by low Mg(2+) concentrations. By supplying additional Mg(2+), site-specific integration occurs in up to 38% of plasmid target sites. The integration products isolated from X. laevis nuclei are sensitive to restriction enzymes specific for double-stranded DNA, indicating second-strand synthesis via host enzymes. We also show that group II intron RNPs containing either lariat or linear intron RNA can introduce a double-strand break into a plasmid target site, thereby stimulating homologous recombination with a co-transformed DNA fragment at frequencies up to 4.8% of target sites. Chromatinization of the target DNA inhibits both types of targeting reactions, presumably by impeding RNP access. However, by using similar RNP microinjection methods, we show efficient Mg(2+)-dependent group II intron integration into plasmid target sites in zebrafish (Danio rerio) embryos and into plasmid and chromosomal target sites in Drosophila melanogster embryos, indicating that DNA replication can mitigate effects of chromatinization.Our results provide an experimental foundation for the development of group II intron-based gene targeting methods for higher organisms

Group II intron mobility and its gene targeting applications in prokaryotes and eukaryotes

textMobile group II introns are retroelements that insert site-specifically into DNA target sites by a process called retrohoming. Retrohoming is mediated by a ribonucleoprotein particle (RNP) that contains both the intron RNA and the intronencoded protein (IEP). My dissertation focuses on two mobile group II introns: Lactococcus lactis Ll.LtrB and Escherichia coli EcI5, which belong to structural subclasses IIA and CL/IIB1, respectively. Previous studies showed that the Ll.LtrB IEP, denoted LtrA protein, is pole localized in E. coli. First, I found that active LtrA protein is associated with E. coli membrane fractions, suggesting that LtrA pole localization might reflect association with a membrane receptor. Second, I found that EcI5 is highly active in retrohoming in E. coli and obtained a comprehensive view of its DNA target site recognition by selection experiments. I found that EcI5 recognizes DNA target sequences by using both the IEP and base pairing of the intron RNA, with the IEP having different target specificity than for other mobile group II introns. A computer algorithm based on the empirically determined DNA recognition rules enabled retargeting of EcI5 to integrate at ten different sites in the chromosomal lacZ gene at frequencies up to 98% without selection. Finally, I developed methods for gene targeting in the frog Xenopus laevis by using Ll.LtrB RNPs for site-specific DNA modification in isolated sperm nuclei, followed by in vitro fertilization to generate genetically modified animals. The site-specific integrations were efficient enough to detect in fifty sperm nuclei for a multiple copy target site, the Tx1 transposon, and several hundred sperm nuclei for protein-encoding genes. Based on these results, I obtained transgenic tadpoles with sitespecific Tx1 integrations by simple screening. To facilitate screening for embryos with targeted integrations in protein-encoding genes, I constructed an intron carrying a GFPRAM (Retrotransposition-Activated Marker). By using this GFP-RAM with introns containing randomized sequences that base pair with the target DNA, I obtained tadpoles with intron integrations at different genomic locations, including protein-encoding genes. The methods for using group II introns for targeted sperm DNA modification in X. laevis may be applicable to other animals.Microbiolog

Makkuni JayaramGroup II Intron Mobility and its Gene Targeting Applications in

b

Small RNA Expression Profiling by High-Throughput Sequencing: Implications of Enzymatic Manipulation

Eukaryotic regulatory small RNAs (sRNAs) play significant roles in many fundamental cellular processes. As such, they have emerged as useful biomarkers for diseases and cell differentiation states. sRNA-based biomarkers outperform traditional messenger RNA-based biomarkers by testing fewer targets with greater accuracy and providing earlier detection for disease states. Therefore, expression profiling of sRNAs is fundamentally important to further advance the understanding of biological processes, as well as diagnosis and treatment of diseases. High-throughput sequencing (HTS) is a powerful approach for both sRNA discovery and expression profiling. Here, we discuss the general considerations for sRNA-based HTS profiling methods from RNA preparation to sequencing library construction, with a focus on the causes of systematic error. By examining the enzymatic manipulation steps of sRNA expression profiling, this paper aims to demystify current HTS-based sRNA profiling approaches and to aid researchers in the informed design and interpretation of profiling experiments

Bias in Ligation-Based Small RNA Sequencing Library Construction Is Determined by Adaptor and RNA Structure

<div><p>High-throughput sequencing (HTS) has become a powerful tool for the detection of and sequence characterization of microRNAs (miRNA) and other small RNAs (sRNA). Unfortunately, the use of HTS data to determine the relative quantity of different miRNAs in a sample has been shown to be inconsistent with quantitative PCR and Northern Blot results. Several recent studies have concluded that the major contributor to this inconsistency is bias introduced during the construction of sRNA libraries for HTS and that the bias is primarily derived from the adaptor ligation steps, specifically where single stranded adaptors are sequentially ligated to the 3’ and 5’-end of sRNAs using T4 RNA ligases. In this study we investigated the effects of ligation bias by using a pool of randomized ligation substrates, defined mixtures of miRNA sequences and several combinations of adaptors in HTS library construction. We show that like the 3’ adaptor ligation step, the 5’ adaptor ligation is also biased, not because of primary sequence, but instead due to secondary structures of the two ligation substrates. We find that multiple secondary structural factors influence final representation in HTS results. Our results provide insight about the nature of ligation bias and allowed us to design adaptors that reduce ligation bias and produce HTS results that more accurately reflect the actual concentrations of miRNAs in the defined starting material.</p></div

Improvement of 5’ adaptor ligation efficiency with designed adaptors.

<p>(A) Scheme depicting a miRNA (blue) attached to a 3’ A1 adaptor (brown) and designed 5’ adaptors that have a region complementary to the miRNA sequence (designed; red) or the 3’ adaptor sequence (C3; violet). (B,C) Comparison of 5’ ligation reaction products for four different miRNAs. Each ligation reaction contained a miRNA-3’ A1 hybrid and either the 5’ A1 adaptor, designed 5’ adaptor (B) or C3 5’ adaptor (C), ATP and T4 RNA ligase 1. The reaction products were resolved using an Agilent Small RNA kit and an Agilent 2100 Bioanalyzer (Agilent Technologies).</p

HTS results from libraries made using different oligonucleotide mixtures.

<p>Three oligonucleotide mixtures were subjected to HTS using different adaptors, as indicated. The adaptors used in library construction are represented by solid lines (defined sequence), jagged lines (randomized sequence), and a solid line with a gray background (region that is complementary to the 5’-end of the 3’ adaptor) and their sequences can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126049#pone.0126049.s006" target="_blank">S1 Table</a>. These oligonucleotide mixtures each contained the same 50 sequences, but the sequences were mixed in different amounts; either equivalent or different 500-fold spreads in concentration (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126049#pone.0126049.s011" target="_blank">S6 Table</a>). Normalized reads for the 50 miRNA sequences in each data set are presented as individual data points on a logarithmic scale. Each miRNA’s normalized reads value was divided by its expected value given the concentration of the miRNA in the defined mix. A result of “1” after this division indicates that the normalized reads were equal to the expected value (dark gray line). The interval of 2-fold from the expected value (equivalent to a 2-fold over or under representation) is shown with light gray lines and 10-fold under the expected value is shown with a red line. The percentage of data points that are >10-fold under the expected value is shown next to a vertical red bar, and the percentage <2-fold from the expected value is shown next to a vertical green bar. The percentages of data points in other regions are shown next to vertical yellow bars. The Mann-Whitney test was used to compare each IT MidRand data set to its corresponding A1 adaptor data set to determine the statistical significance of the difference between the two sets. Two-tailed p values are indicated as (****) p < 0.0001. The values used to make the graph can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126049#pone.0126049.s011" target="_blank">S6 Table</a>.</p