How Does the Nuclear Run on Assay Work?

Introduction on Nuclear Run-On Assay

A nuclear run-on assay was initially developed as a method to establish the transcription rate that plays a part in the regulated expression of mammalian genes. It is used to identify genes that are transcribed at specific times. About a million cell nuclei are isolated, then incubated with labeled nucleotides. Genes that are being transcribed are thus detected through the hybridization of extracted RNA to gene specific probes. 

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The difference of monitored gene expression through nuclear run-on assay versus other assays is that the nuclear run-on assay offers a measure of the frequency of transcription initiation while being largely independent of the effects of RNA stability. This is important as RNAs are known for its instability if not properly extracted. 

For example, one research team that developed a protocol for the yeast S. cerevisiae was able to calculate the transcription rates for all yeast genes to estimate mRNA stabilities for all yeast mRNAs. Hybridization assays provide a measure of how much mRNA is present known as the “steady-state” level as it reflects the balance between mRNA synthesis and degradation while transcription assays provide a direct measure of gene activity.

Gene Expression

The total process of gene expression may be considered to comprise of all the factors that influence the amount of a particular gene product’s presence at a specific time in an organism’s development, under the influence of both endogenous and exogenous effects. There are many steps to this process that includes:

·         Signal transduction

·         Transcriptional level controls

·         Posttranscriptional effects on mRNA level

·         Translational controls

·         Protein turnover

However, it is the gene transcription that is the most crucial step in the entire process. Since the direct measure of transcription rates of specific genes in higher organisms are hard to measure, usually measurements of steady state levels of mRNA species are often considered to be equivalent to gene expression. However, the production and maintenance of an mRNA of a species is subjected to many different factors as its synthesis is determined not only by transcription but also by post-transcriptional processing. the steady state level reflects both the rates of synthesis and degradation. If there are methods that could measure the rates of the different processes, comparison rates of mRNA synthesis and degradation along with measurements of steady stale levels of mRNAs in nuclear and cytoplasmic or total RNA would allow conclusions to be drawn regarding the points of regulation.

There are multiple assays for different purposes. For example:

  • In vivo assays for transcriptional activity of specific genes: a simple approach used to measure the rates of synthesis and degradation of a specific mRNA by measuring the time course of changing mRNA levels after the system is stimulated to cause a change in the steady state levels of mRNA.
  • Assays for transcriptional activity of specific genes using isolated nuclei: Isolated nuclei is used to generate labeled transcripts. This has significant advantage over the use of whole cells as it is able to generate highly labeled RNA.

Alternative methods

Alternative microarray has been recently developed. For example, the PolII RIP-chip: RNA immunoprecipitation of RNA polymerase II with phosphorylated C-terminal domain directed antibodies and hybridization on a microarray slide or chip. A comparison between methods based on run-on and ChIP-chip has been made using yeast where it was concluded that run-on only detects elongating RNA polymerases while ChIP-chip detects all RNA polymerases that are present including those that are backtracked. The attachment of new RNA polymerase to genes are prevented when there is inclusion of Sarkosyl. Hence, only genes that already have a RNA polymerase will be able to produce labeled transcripts. Transcripts synthesized before addition of the label cannot be detected as they lack the label. By purifying labeled transcripts using antibodies that detect the label, and hybridizing isolated transcripts with gene expression arrays, the run on transcripts can also be detected.

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Run on assays have mostly been replaced by Global Run on assays that utilize next generation DNA sequencing as a readout platform. Known as next generation sequencing or GRO-Seq, they provide a much-detailed view of genes that are engaged in transcription while also providing quantitative levels of expression. Array methods that are based on analysis of Global run on (GRO) assays are supplanted by Next Generation Sequencing which removes the design of probes against gene sequences. All transcripts will be catalogued through sequencing even if it is not reported in the database. New transcripts are labeled with bromouridine (BrU) while cells or nuclei incubated with BrUTP with Sarkosyl will prevent the attachment of RN polymerase to DNA. Hence, only RNA polymerase that are already available on the DNA before Sarkosyl is added will be able to produce new transcripts that is labeled with BrU. Labeled transcripts are then captured using anti-BrU antibody beads, converted to cDNAs, and eventually sequenced using the Next Generation DNA sequencing. Lastly, the sequencing reads are aligned according to genome and number of reads per transcript so that an accurate approximation of the number of transcripts synthesized can be made.

References:

Gatehouse G, Thompson AJ. Nuclear “Run-on” transcription assays.

Nuclear run-on. Wikipedia. https://en.wikipedia.org/wiki/Nuclear_run-on