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14.3: The Biology of mRNA Sequencing - Biology

14.3: The Biology of mRNA Sequencing - Biology


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The first step in mRNA sequencing is to lyse the cells of interest. After establishing these sequenced reads, the computational part of RNA-Seq can be divided into three parts: read mapping, reconstruction, and quantification.


14.3 Basics of DNA Replication

By the end of this section, you will be able to do the following:

  • Explain how the structure of DNA reveals the replication process
  • Describe the Meselson and Stahl experiments

The elucidation of the structure of the double helix provided a hint as to how DNA divides and makes copies of itself. In their 1953 paper, Watson and Crick penned an incredible understatement: "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." With specific base pairs, the sequence of one DNA strand can be predicted from its complement. The double-helix model suggests that the two strands of the double helix separate during replication, and each strand serves as a template from which the new complementary strand is copied. What was not clear was how the replication took place. There were three models suggested (Figure 14.12): conservative, semi-conservative, and dispersive.

In conservative replication, the parental DNA remains together, and the newly formed daughter strands are together. The semi-conservative method suggests that each of the two parental DNA strands acts as a template for new DNA to be synthesized after replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. In the dispersive model, both copies of DNA have double-stranded segments of parental DNA and newly synthesized DNA interspersed.

Meselson and Stahl were interested in understanding how DNA replicates. They grew E. coli for several generations in a medium containing a “heavy” isotope of nitrogen ( 15 N), which gets incorporated into nitrogenous bases, and eventually into the DNA (Figure 14.13).

The E. coli culture was then placed into medium containing 14 N and allowed to grow for several generations. After each of the first few generations, the cells were harvested and the DNA was isolated, then centrifuged at high speeds in an ultracentrifuge. During the centrifugation, the DNA was loaded into a gradient (typically a solution of salt such as cesium chloride or sucrose) and spun at high speeds of 50,000 to 60,000 rpm. Under these circumstances, the DNA will form a band according to its buoyant density: the density within the gradient at which it floats. DNA grown in 15 N will form a band at a higher density position (i.e., farther down the centrifuge tube) than that grown in 14 N. Meselson and Stahl noted that after one generation of growth in 14 N after they had been shifted from 15 N, the single band observed was intermediate in position in between DNA of cells grown exclusively in 15 N and 14 N. This suggested either a semi-conservative or dispersive mode of replication. The DNA harvested from cells grown for two generations in 14 N formed two bands: one DNA band was at the intermediate position between 15 N and 14 N, and the other corresponded to the band of 14 N DNA. These results could only be explained if DNA replicates in a semi-conservative manner. And for this reason, therefore, the other two models were ruled out.

During DNA replication, each of the two strands that make up the double helix serves as a template from which new strands are copied. The new strands will be complementary to the parental or “old” strands. When two daughter DNA copies are formed, they have the same sequence and are divided equally into the two daughter cells.


The emerging biology of RNA post-transcriptional modifications

RNA modifications have long been known to be central in the proper function of tRNA and rRNA. While chemical modifications in mRNA were discovered decades ago, their function has remained largely mysterious until recently. Using enrichment strategies coupled to next generation sequencing, multiple modifications have now been mapped on a transcriptome-wide scale in a variety of contexts. We now know that RNA modifications influence cell biology by many different mechanisms - by influencing RNA structure, by tuning interactions within the ribosome, and by recruiting specific binding proteins that intersect with other signaling pathways. They are also dynamic, changing in distribution or level in response to stresses such as heat shock and nutrient deprivation. Here, we provide an overview of recent themes that have emerged from the substantial progress that has been made in our understanding of chemical modifications across many major RNA classes in eukaryotes.

Keywords: Epitranscriptome RNA modification gene expression post-transcriptional regulation protein translation.

Figures

Chemical structures of RNA modifications…

Chemical structures of RNA modifications currently characterized in mRNA, schematized with their reported…


Contents

The brief existence of an mRNA molecule begins with transcription, and ultimately ends in degradation. During its life, an mRNA molecule may also be processed, edited, and transported prior to translation. Eukaryotic mRNA molecules often require extensive processing and transport, while prokaryotic mRNA molecules do not. A molecule of eukaryotic mRNA and the proteins surrounding it are together called a messenger RNP.

Transcription

Transcription is when RNA is copied from DNA. During transcription, RNA polymerase makes a copy of a gene from the DNA to mRNA as needed. This process differs slightly in eukaryotes and prokaryotes. One notable difference is that prokaryotic RNA polymerase associates with DNA-processing enzymes during transcription so that processing can proceed during transcription. Therefore, this causes the new mRNA strand to become double stranded by producing a complementary strand known as the tRNA strand, which when combined are unable to form structures from base-pairing. Moreover, the template for mRNA is the complementary strand of tRNA, which is identical in sequence to the anticodon sequence that the DNA binds to. The short-lived, unprocessed or partially processed product is termed precursor mRNA, or pre-mRNA once completely processed, it is termed mature mRNA.

Eukaryotic pre-mRNA processing

Processing of mRNA differs greatly among eukaryotes, bacteria, and archaea. Non-eukaryotic mRNA is, in essence, mature upon transcription and requires no processing, except in rare cases. [2] Eukaryotic pre-mRNA, however, requires several processing steps before its transport to the cytoplasm and its translation by the ribosome.

Splicing

The extensive processing of eukaryotic pre-mRNA that leads to the mature mRNA is the RNA splicing, a mechanism by which introns or outrons (non-coding regions) are removed and exons (coding regions) are joined together.

5' cap addition

A 5' cap (also termed an RNA cap, an RNA 7-methylguanosine cap, or an RNA m 7 G cap) is a modified guanine nucleotide that has been added to the "front" or 5' end of a eukaryotic messenger RNA shortly after the start of transcription. The 5' cap consists of a terminal 7-methylguanosine residue that is linked through a 5'-5'-triphosphate bond to the first transcribed nucleotide. Its presence is critical for recognition by the ribosome and protection from RNases.

Cap addition is coupled to transcription, and occurs co-transcriptionally, such that each influences the other. Shortly after the start of transcription, the 5' end of the mRNA being synthesized is bound by a cap-synthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions that are required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction.

Editing

In some instances, an mRNA will be edited, changing the nucleotide composition of that mRNA. An example in humans is the apolipoprotein B mRNA, which is edited in some tissues, but not others. The editing creates an early stop codon, which, upon translation, produces a shorter protein.

Polyadenylation

Polyadenylation is the covalent linkage of a polyadenylyl moiety to a messenger RNA molecule. In eukaryotic organisms most messenger RNA (mRNA) molecules are polyadenylated at the 3' end, but recent studies have shown that short stretches of uridine (oligouridylation) are also common. [3] The poly(A) tail and the protein bound to it aid in protecting mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. mRNA can also be polyadenylated in prokaryotic organisms, where poly(A) tails act to facilitate, rather than impede, exonucleolytic degradation.

Polyadenylation occurs during and/or immediately after transcription of DNA into RNA. After transcription has been terminated, the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase. After the mRNA has been cleaved, around 250 adenosine residues are added to the free 3' end at the cleavage site. This reaction is catalyzed by polyadenylate polymerase. Just as in alternative splicing, there can be more than one polyadenylation variant of an mRNA.

Polyadenylation site mutations also occur. The primary RNA transcript of a gene is cleaved at the poly-A addition site, and 100–200 A's are added to the 3’ end of the RNA. If this site is altered, an abnormally long and unstable mRNA construct will be formed.

Transport

Another difference between eukaryotes and prokaryotes is mRNA transport. Because eukaryotic transcription and translation is compartmentally separated, eukaryotic mRNAs must be exported from the nucleus to the cytoplasm—a process that may be regulated by different signaling pathways. [4] Mature mRNAs are recognized by their processed modifications and then exported through the nuclear pore by binding to the cap-binding proteins CBP20 and CBP80, [5] as well as the transcription/export complex (TREX). [6] [7] Multiple mRNA export pathways have been identified in eukaryotes. [8]

In spatially complex cells, some mRNAs are transported to particular subcellular destinations. In mature neurons, certain mRNA are transported from the soma to dendrites. One site of mRNA translation is at polyribosomes selectively localized beneath synapses. [9] The mRNA for Arc/Arg3.1 is induced by synaptic activity and localizes selectively near active synapses based on signals generated by NMDA receptors. [10] Other mRNAs also move into dendrites in response to external stimuli, such as β-actin mRNA. [11] Upon export from the nucleus, actin mRNA associates with ZBP1 and the 40S subunit. The complex is bound by a motor protein and is transported to the target location (neurite extension) along the cytoskeleton. Eventually ZBP1 is phosphorylated by Src in order for translation to be initiated. [12] In developing neurons, mRNAs are also transported into growing axons and especially growth cones. Many mRNAs are marked with so-called "zip codes," which target their transport to a specific location. [13]

Translation

Because prokaryotic mRNA does not need to be processed or transported, translation by the ribosome can begin immediately after the end of transcription. Therefore, it can be said that prokaryotic translation is coupled to transcription and occurs co-transcriptionally.


Using mRNA to develop a new category of medicines.

At Moderna, we are leveraging the fundamental role that mRNA plays in protein synthesis. We have developed proprietary technologies and methods to create mRNA sequences that cells recognize as if they were produced in the body. We focus on diseases where enabling targeted cells to produce – or turn ‘on’ – one or more given proteins will enable the body to fight or prevent a given disease.

  • We start with our desired sequence for a protein.
  • We design and synthesize the corresponding mRNA sequence – the code that will create that protein.
  • Before synthesis, we also engineer that mRNA sequence to optimize the mRNA’s physical properties, as well as those of the encoded protein.
  • We deliver the mRNA sequence to the cells responsible for making that protein via one of several modalities. Reaching different types of cells requires different delivery methods.
  • And, once the mRNA – the instructions – are in the cell … human biology takes over. Ribosomes read the code and build the protein, and the cells express the protein in the body.

Using mRNA as a drug opens up a breadth of opportunities to treat and prevent disease. mRNA medicines can go inside cells to direct protein production, something not possible with other drug approaches. We have the potential to treat or prevent diseases that today are not addressable – potentially improving human health and impacting lives around the world.

Learn about the intrinsic features of mRNA, how it is used in cells throughout the body and the diversity of potential applications for using mRNA to develop new medicines.


Watch the video: Illumina mRNA sequencing (May 2022).