分子生物学

Fork stability is key to genome DNA duplication and genetic integrity. Long non-coding RNAs (LncRNAs) may play vital roles in fork stabilization and chromatin remodeling. Existing techniques such as NCC-RNA sequencing are useful to identify LncRNAs on nascent chromatin DNA. However, there is still a lack of methods for LncRNAs purification directly from replicative forks, hindering a deep understanding of the functions of LncRNAs in fork regulation. Here, we provide a step-by-step protocol named iROND (isolate RNAs on nascent DNA). iROND was developed and modified from iPOND, a well-known method for purifying fork-associated proteins. iROND relies on click chemistry reaction of 5'-ethynyl-2'-deoxyuridine (EdU)-labeled forks and biotin. After streptavidin pull down, fork-associated LncRNAs and proteins are purified simultaneously. iROND is compatible with downstream RNA sequencing, qPCR confirmation, and immunoblotting. Integrated with functional methods such as RNA fluorescent in situ hybridization (RNA FISH) and DNA fiber assay, it is feasible to screen fork-binding LncRNAs in defined cell lines and explore their functions. In summary, we provide a purification pipeline of fork-associated LncRNAs. iROND is also useful for studying other types of fork-associated non-coding RNAs.

Key features

• Purify long non-coding RNAs (LncRNAs) directly from replication forks.

• Connects to RNA sequencing for screening easily.

• Allows testing various genotoxic stress responses.

• Provides LncRNA candidate list for downstream functional research.

Graphical overview

Schematic overview of isolate RNAs on nascent DNA (iROND) protocol. Cells were pulse-labeled with 5'-ethynyl-2'-deoxyuridine (EdU) for 10 min before paraformaldehyde fixation. EdU-positive forks were ligated with biotin through Click-IT chemistry reaction. Genomic DNA was ultrasonically cracked and crosslinked with streptavidin for pulling down. Both RNA and protein components were purified. RNA components were used for downstream RNA sequencing and qPCR validation. Protein components were used for immunoblotting to evaluate binding dynamics of fork-associated proteins such as helicase, topoisomerase, and DNA polymerases.

RNA-RNA and RNA-protein interactions are involved in the regulation of gene expression. Here, we describe an updated and extended version of our RNA purification and protein identification (RaPID) protocol for the pulldown of aptamer-tagged mRNAs by affinity purification. The method takes advantage of the high affinity interaction between the MS2 RNA aptamer and the MS2 coat protein (MCP), as well as that between streptavidin-binding peptide (SBP) and streptavidin. Thus, it employs MCP-SBP fusions to affinity purify MS2-tagged target RNAs of interest over immobilized streptavidin. Purified aptamer-tagged mRNAs, along with any associated RNAs and proteins, are then sent for RNA sequencing (RaPID-seq) or mass spectrometry (RaPID-MS), which allows for the identification of bound cohort RNAs and proteins, respectively.

Transfer RNAs (tRNAs) are heavily decorated with post-transcriptional modifications during their biosynthesis. To fulfil their functions within cells, tRNAs undergo a tightly controlled biogenesis process leading to the formation of mature tRNAs. In addition, functions of tRNAs are often modulated by their modifications. Although the biological importance of post-transcriptional RNA modifications is widely appreciated, methods to directly detect their introduction during RNA biosynthesis are rare and do not easily provide information on the temporal nature of events. To obtain information on the tRNA maturation process, we have developed a methodology, using NMR as a tool to monitor tRNA maturation in a non-disruptive and continuous fashion in cellular extracts. By following the maturation of a model yeast tRNA with time-resolved NMR, we showed that modifications are introduced in a defined sequential order, and that the chronology is controlled by cross-talk between modification events. The implementation of this method requires the production for NMR spectroscopy of tRNA samples with different modification status, in order to identify the NMR signature of individual modifications. The production of tRNA samples for the analysis of modification pathways with NMR spectroscopy will be presented here and examplified on the yeast tRNA^Phe, but can be extended to any other tRNA by changing the sequence of the construct. The protocol describes the production of unmodified tRNA samples by in vitro transcription, and the production of modified tRNA samples by recombinant expression of tRNAs in E. coli.

Cell type-specific molecular profiling is widely used to gain new insights into the diverse cell types that make up complex biological tissues. Translating ribosome affinity purification (TRAP) is a method in which the cell type-specific expression of epitope-tagged ribosomal subunits allows one to purify actively translating mRNAs without the need for cell sorting or fixation. We adapted this method for use in C. elegans to identify novel transcripts in single cell types or to identify the effects of environmental changes on the transcriptomes of larger cohorts of cells. In this protocol, we describe methods to generate transgenic animals bearing tagged ribosomes in cells of interest, prepare these animals for immunoprecipitation, purify ribosome-mRNA complexes, and obtain purified mRNA for next-generation sequencing.

The early embryo of Drosophila melanogaster exists as a rapidly dividing syncytium of nuclei that are transcriptionally silent. Maternally deposited factors are required to awaken the genome and assist in the transition from maternal to zygotic control of development. Because many of these essential factors are maternally deposited and the early nuclear divisions are so rapid, it has been difficult to assess the functional role of transcription factors at discrete points in early embryonic development. To address this issue, we have developed an optogenetic system that can rapidly and reversibly inactivate transcription factors with nuclear-cycle resolution. The temporal precision enabled by this technique will allow a mechanistic understanding of how transcription factors function together to control genome activation and patterning in the early embryo and is likely broadly applicable to factors throughout embryogenesis.

In virology the difference between the fitness of two viruses can be determined by using various methods, such as virus titer, growth curve analysis, measurement of virus infectivity, analysis of produced RNA copies and viral protein production. However, for closely performing viruses, it is often very hard to distinguish the differences. In vitro competition assays are a sensitive tool for determining viral replication fitness for many viruses replicating in cell culture. Relative viral replication fitness is usually measured from multiple cycle growth competition assays. Competition assays provide a sensitive measurement of viral fitness since the viruses are competing for cellular targets under identical growth conditions. This protocol describes a competition assay for enteroviruses and contains two alternative formats for initial infections, which can be varied depending on specific goals for each particular experiment. The protocol involves infection of cells with competing viruses, passaging, RNA extraction from infected cells, RT-PCR and Sanger sequencing followed by comparative analysis of resulting chromatograms obtained under various initial infection conditions. The techniques are applicable to members of many virus families, such as alphaviruses, flaviviruses, pestiviruses, and other RNA viruses with an established reverse genetics system.

Long Interspersed Nuclear Element-1 (LINE-1, L1) constitutes a family of autonomous, self-replicating genetic elements known as retrotransposons. Although most are inactive, copious L1 sequences populate the human genome. L1s proliferate in a ‘copy-and-paste’ fashion through an RNA intermediate; a full-length L1 transcript is ~6,000 nucleotides long and functions as a bicistronic mRNA that encodes and assembles in cis with two main polypeptides, ORF1p and ORF2p, forming a ribonucleoprotein (RNP); L1 RNPs also interact with a wide range of host factors in positive and negative regulatory capacities. The following protocol describes an approach to affinity enrich ectopically expressed L1 RNPs and, using RNases, release the fraction of protein that depends upon the presence of intact RNA for retention in the immobilized macromolecules.