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What does it mean that the transcript is enriched?

What does it mean that the transcript is enriched?


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I think I don't get the meaning of "enriched" in the context of genes. What's the difference of gene being "enriched" and "expressed" in the cell?


In most cases (i.e. assuming there is no aneuploidy) a gene won't be enriched, however, the transcript from a gene may be enriched. A synonym in this case would be overrepresented. In other words, you find relatively more of the transcript compared to other transcripts.

Since you seem to confused about the difference between transcripts and genes, I highly recommend working through the material on a site like Khan Academy. This will help you understand the basic vocabulary and concepts of molecular biology. There are also a number free online textbooks available at NCBI where you can get more in depth information.


In the context of transcriptomics the term 'enrichment' is usually connected to differential analysis:

  • If a transcript (or some/all transcripts of a gene) are detected in a given sample that transcript is expressed
  • If a transcript is detected at (statistically significant) higher levels in sample (or condition) A compared to another sample B, that transcript is enriched in sample A.

In some cases one might compare two cell types, where only one expresses a given transcript, in this expression and enrichment in that cell line (compared to the other one) would be almost the same.


4.5: Transcription of DNA to RNA

  • Contributed by CK-12: Biology Concepts
  • Sourced from CK-12 Foundation

How does a cell use the information in its DNA?

To transcribe means &lsquo&lsquoto paraphrase or summarize in writing&rsquo&rsquo. The information in DNA is transcribed - or summarized - into a smaller version - RNA - that can be used by the cell. This process is called transcription.


Transcription Factors

Transcription Factors: What They Do

Transcription factors play many different roles, which vary according to the organism in question. For example, in vertebrates, transcription factors are directly responsible for development, with groups of different factors coming into play in specific tissues. Transcription factors are especially important during embryonic development and thus specific factors are essential for the differentiation of pluripotent embryonic stem cells. Similarly, the activity of other factors must be maintained for stem cells to retain their ability to turn into any cell type and to self-renew. It is not surprising that many human diseases or abnormalities are caused by the misfunction of transcription factors. Similarly, somatic mutation or chromosomal rearrangements that affect certain transcription factors play a key role in the development of some human cancers. Understanding how the sequential deployment of transcription factors controls differentiation and development is a vibrant current area of research and it is important to note the value of studies with mice, zebra fish, fruit flies, and nematodes in understanding how transcription factors drive development. The situation in unicellular organisms is different where the primary role of transcription factors is to manage adaptation to environmental change, for example, sensing nutrients or coping with life in stressful niches. Detailed information on the number and nature of transcription factors in different organisms can be found on many websites.


Ribosome Structure and Function

Ribosomes are macromolecular, multi-subunit structures containing RNA as well as protein and are the primary machines that drive protein synthesis. The structure of the ribosome derives primarily from it RNA component (ribosomal RNA or rRNA) and base-pairing with mRNA and tRNA is crucial to its function.

The ribosome contains two subunits and translation is initiated when the smaller subunit binds to sequences upstream of the coding sequence on the mRNA. Prokaryotic translation begins with the rRNA directly binding to the mRNA, whereas eukaryotic translation involves other proteins called initiation factors. The smaller subunit, along with some other proteins recruit the larger subunit of the ribosome, and translation begins.

Primarily, the ribosome contains three important regions – the P site, the A site and the E site – formed by the three-dimensional shape of the rRNA. The P site binds to the growing polypeptide, the A site anchors an incoming charged tRNA and after peptide bond formation, the tRNA binds briefly to the E site before leaving the ribosome.


What Is the Relationship Between Transcription and Translation?

Transcription is the process of making an RNA copy of a gene sequence. Translation is the process of translating the sequence of a messenger RNA molecule to a sequence of amino acids during protein synthesis. So, the relationship between the two processes is that they are both involved in protein synthesis and that transcription is first, then translation is second. Ultimately, this is all we know about transcription and translation in terms of genetics. Read on if you want to understand more about transcription and translation when we talk about services.

Whether it&rsquos a public speech, interview, market research or financial report, if you hope for your message to reach an audience as wide as possible, you should consider transcribing and then translating it to your target languages. So, here we go!

The Transcription

Just like in genetics, transcription is necessary to happen first. It is, according to Merriam Webster, the act of making a written, printed, or typed copy of words that have been spoken. Having a documented text format of your audio and video files is utterly necessary for businesses, for example. It is a pragmatic way to keep track and understand better what is being said. If you liked the idea, plan carefully as it takes lots of time for anyone inexperienced in transcription to turn an audio record into a clear text document. Tip: leave this tiring and tedious task to our professional transcriptionists who guarantee confidentiality, accuracy and the highest of quality.

The Translation

Once you have a ready and proofread transcript, you can proceed further with translation. It is basically the act of changing one language into another. However, in practice it is not simply changing words into their equivalents in different languages. Quality translation involves knowing the context and cultural background from which the words in the original text came, and then choosing words and phrases in the new language which will best convey the substance and meaning of the original in a new and different cultural context. Unless all this happens, the message may get misinterpreted or even lost. Moreover, if you are going to invest resources for this service, make sure you seek for the best timing, simplicity and audience adaptation.


Enrich

We’ve also learned that by making the crowd smaller, we really are enrich ing the visitor experience.

The same month, Smith and his partners sold a minority stake in Vista Equity Partners to another private equity firm, a move that likely would have enrich ed him, better enabling him to become a donor.

Throughout the book, secondary players help enrich Cheney’s story.

High Place claims that it provides El Triunfo with jobs and enrich es the economy, but that’s just a pretty lie.

“Engagement Insights is about directly funneling web, mobile and connected product data back into Customer Insights to help continue to enrich that understanding of the customer in order to better serve them,” he said.

Importantly, as part of the interim plan, Iran has diluted or converted its stockpile of 20 percent enrich ed uranium.

The claim is that they “unjustly enrich ed” themselves while damaging the image of the U.S. Postal Service.

“That provides Iran another option to keep their [highly enrich ed uranium] program advancing,” he said.

Tehran is also developing advanced centrifuge designs and stockpiling low- enrich ed uranium.

As we gather here tonight, Iran has begun to eliminate its stockpile of higher levels of enrich ed uranium.

The windows are ornamented by tracery, and the façade is enrich ed by a free use of carving.

As a net is full of birds, so their houses are full of deceit: therefore are they become great and enrich ed.

This enrich ed the organ with a new group of stops of a superior quality on account of the roundness and volume of sound.

A standard and authoritative work enrich ed by copious illustrations.

All around her was the wide awe of night, enrich ed by the sweet perfume of a coming harvest.


Transcription in Eukaryotes | Genetics

Transcription has been defined in various ways. Some definitions of transcription are given here. The synthesis of RNA from a single strand of a DNA molecule in the presence of enzyme RNA polymerase is called transcription. In other words, the process of formation of a messenger RNA molecule using a DNA molecule as a template is referred to as transcription.

The main points related to transcription in eukaryotes are briefly discussed below:

RNA is synthesized from a DNA template. The RNA is processed into messenger RNA [mRNA], which is then used for synthesis of a protein. The RNA thus synthesized is called messenger RNA (mRNA), because it carries a genetic message from the DNA to the protein- synthesizing machinery of the cell.

The main difference between RNA and DNA sequence is the presence of U, or uracil in RNA instead of the T, of thymine of DNA.

The RNA is synthesized from a single strand or template of a DNA molecule. The stretch of DNA that is transcribed into an RNA molecule is called a transcription unit. A transcription unit codes the sequence that is translated into protein. It also directs and regulates protein synthesis.

The DNA strand which is used in RNA synthesis is called template strand because it provides the template for ordering the sequence of nucleotides in an RNA transcript. The DNA strand which does not take part in DNA synthesis is called coding strand, because, its nucleotide sequence is the same as that of the newly created RNA transcript.

The process of transcription is catalyzed by the specific enzyme called RNA polymerase. DNA sequence is enzymatically copied by RNA polymerase to produce a complementary nucleotide RNA strand. In eukaryotes, there are three classes of RNA polymerases: I, II and III which are involved in the transcription of all protein genes.

4. Genetic Information Copied:

In this process, the genetic information coded in DNA is copied into a molecule of RNA. The genetic information is transcribed or copied, from DNA to RNA. In other words, it results in the transfer of genetic information from DNA into RNA.

The expression of a gene consists of two major steps, viz., transcription and translation. Thus transcription is the first step in the process of gene regulation or protein synthesis.

6. Direction of Synthesis:

As in DNA replication, RNA is synthesized in the 5′ —> 3′ direction. The DNA template strand is read 3′ –> 5′ by RNA polymerase and the new RNA strand is synthesized in the 5′ -> 3′ direction. RNA polymerase binds to the 3′ end of a gene (promoter) on the DNA template strand and travels toward the 5′ end.

The regulatory sequence that is before, or 5′, of the coding sequence is called 5′ un-translated region (5′ UTR), and sequence found following, or 3′, of the coding sequence is called 3′ un-translated region (3′ UTR). Transcription has some proofreading mechanisms, but they are fewer and less effective than the controls for copying DNA therefore, transcription has a lower copying fidelity than DNA replication.

Mechanism of Transcription in Eukaryotes:

The mechanism of transcription consists of five major steps, viz:

These are briefly discussed as follows:

1. Pre-Initiation:

The initiation of transcription does not require a primer to start. RNA polymerase simply binds to the DNA and, along with other cofactors, unwinds the DNA to create an initiation bubble so that the RNA polymerase has access to the single-stranded DNA template. However, RNA Polymerase does require a promoter like sequence.

Proximal (core) Promoters:

TATA promoters are found around -30 bp to the start site of transcription. Not all genes have TATA box promoters and there exists TATA-less promoters as well. The TATA promoter consensus sequence is TATA(A/T)A(A/T).

In eukaryotes and archaea, transcription initiation is far more complex. The main difference is that eukaryotic polymerases do not recognize directly their core promoter sequences. In eukaryotes, a collection of proteins called transcription factors mediate the binding of RNA polymerase and the initiation of transcription.

Only after attachment of certain transcription factors to the promoter, the RNA polymerase binds to it. The complete assembly of transcription factors and RNA polymerase bind-to the promoter, called transcription initiation complex. Initiation starts as soon as the complex is opened and the first phosphodiester bond is formed. This is the end of Initiation.

RNA Pol II does not contain a subunit similar to the prokaryotic factor, which can recognize the promoter and unwind the DNA double helix. In eukaryotes, these two functions are carried out by a set of proteins called general transcription factors.

The RNA Pol II is associated with six general transcription factors, designated as TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH, where “TF” stands for “transcription factor” and “II” for the RNA Pol II.

TFIID consists of TBP (TATA-box binding protein) and TAFs (TBP associated factors). The role of TBP is to bind the core promoter. TAFs may assist TBP in this process. In human cells, TAFs are formed by 12 subunits. One of them, TAF250 (with molecular weight 250 kD), has the histone acetyltransferase activity, which can relieve the binding between DNA and histones in the nucleosome.

The transcription factor which catalyzes DNA melting is TFIIH. However, before TFIIH can unwind DNA, the RNA Pol II and at least five general transcription factors (TFIIA is not absolutely necessary) have to form a pre-initiation complex (PIC).

3. Promoter Clearance:

After the first bond is synthesized the RNA polymerase must clear the promoter. During this time there is a tendency to release the RNA transcript and produce truncated transcripts. This is called abortive initiation and is common for both Eukaryotes and Prokaryotes.

Once the transcript reaches approximately 23 nucleotides it no longer slips and elongation can occur. This is an ATP dependent process. Promoter clearance also coincides with Phosphorylation of serine 5 on the carboxy terminal domain which is phosphorylated by TFIIH.

For RNA synthesis, one strand of DNA known as the template strand or non-coding strand is used as a template. As transcription proceeds, RNA polymerase traverses the template strand and uses base pairing complementarity with the DNA template to create an RNA copy.

Although RNA polymeras traverses the template strand from 3′ —> 5′, the coding (non-template) strand is usually used as the reference point, so transcription is said to go from 5′ —> 3′.

This produces an RNA molecule from 5′ —> 3′, an exact copy of the coding strand (except that thymines are replaced with uracils, and the nucleotides are composed of a ribose (5-carbon) sugar where DNA has deoxyribose (one less oxygen atom) in its sugar-phosphate backbone).

After pre-initiation complex [PIC] is assembled at the promoter, TFIIH can use its helicase activity to unwind DNA. This requires energy released from ATP hydrolysis. The DNA melting starts from about -10 bp.

Then, RNA Pol II uses nucleoside triphosphates (NTPs) to synthesize a RNA transcript. During RNA elongation, TFIIF remains attached to the RNA polymerase, but all of the other transcription factors have dissociated from PIC.

The carboxyl-terminal domain (CTD) of the largest subunit of RNA Pol II is critical for elongation. In the initiation phase, CTD is un-phosphorylated, but during elongation it has to be phosphorylated. This domain contains many proline, serine and threonine residues.

In eukaryotic transcription the mechanism of termination is not very clear. In other words, it is not well understood. It involves cleavage of the new transcript, followed by template- independent addition of As at its new 3′ end, in a process called polyadenylation.

Eukaryotic protein genes contain a poIy-A signal located downstream of the last exon. This signal is used to add a series of adenylate residues during RNA processing. Transcription often terminates at 0.5-2 kb downstream of the poly-A signal.

Transcription Factories in Eukaryotes:

Active transcription units that are clustered in the nucleus, in discrete sites are called ‘transcription factories’. Such sites could be visualized after allowing, engaged polymerases to extend their transcripts in tagged precursors (Br-UTP or Br-U), and immuno-labelling the tagged nascent RNA.

Transcription factories can also be localized using fluorescence in situ hybridization, or marked by antibodies directed against polymerases. There are

10,000 factories in the nucleoplasm of a HeLa cell, among which are

8,000 polymerase II factories and

2,000 polymerase III factories. Each polymerase II factory contains

As most active transcription units are associated with only one polymerase, each factory will be associated with

8 different transcription units. These units might be associated through promoters and/or enhancers, with loops forming a ‘cloud’ around the factory.

Reverse Transcription in Eukaryotes:

Synthesis of DNA from RNA molecule in the presence of enzyme reverse transcriptase is referred to as reverse transcription. Reverse transcription was first reported by Temin and Baltimore in 1970 for which they were awarded Nobel prize in 1975. Reverse transcription is also known as Teminism. Some viruses (such as HIV, the cause of AIDS), have the ability to transcribe RNA into DNA.

In some eukaryotic cells, an enzyme is found with reverse transcription activity. It is called telomerase. Telomerase is a reverse transcriptase that lengthens the ends of linear chromosomes. Telomerase carries an RNA template from which it synthesizes DNA repeating sequence, or “junk” DNA. This repeated sequence of “junk” DNA is important because every time a linear chromosome is duplicated, it is shortened in length.

With “junk” DNA at the ends of chromosomes, the shortening eliminates some repeated, or junk sequence, rather than the protein-encoding DNA sequence that is further away from the chromosome ends.

Telomerase is often activated in cancer cells to enable cancer cells to duplicate their genomes without losing important protein-coding DNA sequence. Activation of telomerase can be part of the process that allows cancer cells to become immortal.

Role of Transcription Factors in Eukaryotes:

In eukaryotes, the association between DNA and histones prevents access of the polymerase and general transcription factors to the promoter. Histone acetylation catalyzed by HATs can relieve the binding between DNA and histones. Although a subunit of TFIID (TAF250 in human) has the HAT activity, participation of other HATs can make transcription more efficient. The following rules apply to most (but not all).

(i) Binding of activators to the enhancer element recruits HATs to relieve association between histones and DNA, thereby enhancing transcription.

(ii) Binding of repressors to the silencer element recruits histone deacetylases (denoted by HDs or HDACs) to tighten association between histones and DNA.


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What does it mean that the transcript is enriched? - Biology

Figure 1: Electron microscopy image of simultaneous transcription and translation. The image shows bacterial DNA and its associated mRNA transcripts, each of which is occupied by ribosomes. (Adapted from O. L. Miller et al., Science 169:392, 1970.)

Transcription, the synthesis of mRNA from DNA, and translation, the synthesis of protein from mRNA, are the main pillars of the central dogma of molecular biology. How do the speeds of these two processes compare? This question is made all the more interesting as a result of observations like those shown in Figure 1, namely, the existence of the beautiful “Christmas tree” structures observed in E. coli using electron microscopy. These stereotyped structures reflect the simultaneous transcription and translation of the same gene and raise the question of how the relative rates of the two processes compare making such synchronization of these two disparate processes possible.

Figure 2: Back of the envelope calculation comparing the rates of transcription and translation showing they are effectively very similar. nt denotes nucleotides, i.e. bases.

Transcription of RNA in E. coli of both mRNA and the stable rRNA and tRNA, is carried out by ≈1000-10,000 RNA polymerase molecules (BNID 101440) proceeding at a maximal speed of about 40-80 nt/sec as shown in Table 1 (BNID 104900, 104902, 108488). Translation of proteins in E. coli is carried out by ≈10,000-100,000 ribosomes (BNID 101441) and proceeds at a maximal speed of about 20 aa/sec as shown in Table 2 (BNID 100059, 105067, 108490). Interestingly, since every 3 base pairs code for one amino acid, the rates of the two processes are nearly matched as schematically shown in Figure 2 (see also BNID 108487). If translation was faster than transcription, it would cause the ribosome to “collide” with the RNA polymerase in prokaryotes where the two processes can happen concurrently. Such co-transcriptional translation has become textbook material through images such as Figure 1. But recent single-molecule microscopy shows this occurs relatively rarely and most translation is not coupled with transcription in E. coli (S. Bakshi et al., Mol Microbiol. 85:21, 2012). Rather, most translation takes place on mRNA that has already diffused away from the DNA rich nucleoid region to ribosome-rich cytoplasmic regions. The distribution of ribosomes in the cells is further shown in the vignette on “How many ribosomes are in a cell?”. In another twist and turn of the central dogma, it was shown that ribosomes can be important for fast transcription in bacteria (S. Proshkin et al., Science 328:504, 2010). The ribosomes seem to keep RNA polymerase from backtracking and pauses, which can otherwise be quite common for these machines, thus creating a striking reverse coupling between translation and transcription.

Table 1. Transcription rate measured across organisms and conditions. All values measured at 37°C except D. melanogaster measured at 22°C.

Table 2: Translation rate measured across organisms and conditions. All values measured at 37°C except for S. cerevisiae and N. crassa measured at 30°C.

What do the relative rates of transcription and translation mean for the overall time taken from transcription initiation to synthesized protein for a given gene? In bacteria, a one kb gene should take at maximal transcription rate about 1000 nt/80 nt/s ≈ 10s and translation elongation at maximal speed roughly the same. We note that the total time scale is the sum of an elongation time as above and the initiation time, which can be longer in some cases. Recently it was observed that increasing the translation rate, by replacing wobble codons with perfect matching codons, results in errors in folding (P. S. Spencer et al, J. Mol. Biol., 422:328, 2012). This suggests a tradeoff where translation rate is limited by the time needed to allow proper folding of domains in the nascent protein.

Figure 3: Effect of rifampin on transcription initiation. Electron micrographs of E. coli rRNA operons: (A) before adding rifampin, (B) 40 s after addition of rifampin, and (C) 70 s after exposure. After drug treatment, no new transcripts are initiated, but those already initiated are carrying on elongation. In parts (A) and (B) the arrow indicates the site where RNaseIII cleaves the nascent RNA molecule producing 16S and 23S ribosomal subunits. RNA polymerase molecules that have not been affected by the antibiotic are marked by the arrows in part (C). (Adapted from L. S. Gotta et al., J. Bacteriol. 20:6647, 1991.)

How are the rates of these key processes of the central dogma measured? This is an interesting challenge even with today’s advanced technologies. Let’s consider how we might attack this problem. One vague idea might be: “let’s express a GFP and measure the time until it appears”. To see the flaws in such an approach, check out the vignette on “What is the maturation time for fluorescent proteins?” (short answer – minutes to an hour), which demonstrates a mismatch of time scales between the processes of interest and those of the putative readout. The experimental arsenal available in the 1970’s when the answers were first convincingly obtained was much more limited. Yet, in a series of clever experiments, using electron microscopy and radioactive labeling these rates were precisely determined (Miller et al., Science 169:392, 1970 R. Y. Young & H. Bremer, Biochem. J., 152:243, 1975). As will be shown below, they relied on a subtle quantitative analysis in order to tease out the rates.

Measurements on transcription rates were based upon a trick in which transcription initiation was shut down by using the drug rifampin. Though no new transcription events can begin, those that are already under way continue unabated, i.e. rifampin inhibits the initiation of transcription, but not the elongation of RNA transcripts. As a result, this drug treatment effectively begins the running of a stopwatch which times how long since the last transcription process began. By fixing the cells and stopping the transcription process at different times after the drug treatment and then performing electron microscopy, resulting in images like that shown in Figure 3, it was possible to measure the length of RNA polymerase-free DNA. By taking into account the elapsed time since drug treatment the rate at which these polymerases are moving is inferred.

Figure 4: Dynamics of transcription in the fly embryo. (A) Schematic of the experiment showing how a loop in the nascent RNA molecule serves as a binding site for a viral protein that has been fused to GFP. (B) Depending upon whether the RNA loops are placed on the 5’ or 3’ end of the mRNA molecule, the time it takes to begin seeing GFP puncta will be different. The delay time is equal to the length of the transcribed region divided by the speed of the polymerase. (C) Microscopy images showing the appearance of puncta associated with the transcription process for both constructs shown in (B). (D) Distribution of times of first appearance for the two constructs yielding a delay time of 2.2 minutes, from which a transcription rate of 25 nt/s is inferred. Measurements performed at room temperature of 22OC. (adapted from H. G. Garcia, et al., Current Biology, 23, 2140–2145, 2013.)

The measurement of translation rates similarly depended upon finding an appropriate stopwatch, but this time for the protein synthesis process. The crux of the method is the following: start adding labeled amino acid at time zero and follow (“chase” as it is often called) the fraction of labeled protein of mass m as defined by looking at a specific band on a gel. Immediately after the pulse of labeled amino acids one starts to see proteins of mass m with radioactive labeled amino acids on their ends. With time, the fraction of a given protein mass that is labeled will increase as the chains have a larger proportion of their length labeled. After a time τm, depending on the transcript length, the whole chain will be labeled, as these are proteins that began their translation at time zero when the label was added. At this time one observes a change in the accumulation dynamics (when appropriately normalized to the overall labeling in the cell). From the time that elapsed, τm, and by knowing how many amino acids are in a polypeptide chain of mass m it is possible to derive an estimate for the translation rate. There are uncertainties associated with doing this that are minimized by performing this for different protein masses, m, and calculating a regression line over all the values obtained. For a full understanding of the method, the reader will benefit from the original study by Young & Bremer, Biochem. J., 160:185, 1976. It remains as a reliable value for E. coli translation rate to this day. We are not aware of newer methods that give better results.

Figure 5: Distribution of measured transcription elongation rates inferred from relieving transcription inhibition and sequencing all transcripts at later time points. (Adapted from G. Fuchs et al., Genome Bio., 15:5, 2014.)

What are the corresponding rates in eukaryotes? As shown in Tables 1 and 2, transcription in mammalian cells consists of elongation at rates similar to those measured in E. coli (50-100 nt/sec, BNID 105566, 105113, 100662). It is suggested that these stretches of rapid transcription are interspersed with pauses leading to an average rate that is about an order of magnitude slower (≈6 nt/sec, 100661), but some reports do not observe such slowing down (BNID 105565). Recent in-vivo measurements in fly embryos have provided a beautiful real-time picture of the transcription process by using fluorescence to watch the first appearance of mRNA as shown in Figure 4. Recently another approach utilizing the power of sequencing inferred the distribution of transcription elongation rates in a HeLa cell line as shown in Figure 5, showing a range of 30-100 nts/s with a median rate of 60 nts/s (BNID 111027). Remember that in eukaryotes, transcription and translation are spatially segregated, with transcription taking place in the nucleus and translation in the cytoplasm. Introns are excised from transcripts prior to translation taking about 5-10 minutes on average for this process of mRNA splicing (BNID 105568). Though our focus here was on transcript elongation, in some cases the rate limiting process seems to be the initiation of transcription. This is the process in which the RNA polymerase complex is assembled, and the two DNA strands are separated to form a bubble that enables transcription.

Figure 6: Inferring the rate of translation by the ribosome in mouse embryonic stem cells using ribosome profiling. (A) Inhibiting translation initiation followed by inhibition of elongation creates a pattern of ribosome stalling dependent on the time differences and rates of translation. Using modern sequencing techniques this can be quantified genome wide and the translation rate accurately measured for each transcript. (B) Measurement of the translation rate using the methodology indicated schematically in part (A). (Adapted from N. Ingolia et al., Cell, 146:789, 2011.

What about the rates of translation in eukaryotes? In budding yeast the rate is about 2 fold slower than that in bacteria (3-10 aa/s, BNID 107871), but one should note that the “physiological” temperature at which it is measured is 30ºC whereas for E. coli, measurements are at 37ºC. As discussed in the vignette on “How does temperature affect rates and affinities?”, the slower rate is what one would expect based on the general dependence of a factor of 2-3 per 10ºC (Q10 value, BNID 100919). Using the method of ribosome profiling based on high-throughput sequencing and schematically depicted in Figure 6, the translation rate in mouse embryonic stem cells was surveyed for many different transcripts. It was found that the rate is quite constant across proteins and is about 6 amino acids per second (BNID 107952). After several decades of intense investigation and ever more elaborate techniques at our disposal we seem to have arrived at the point where the quantitative description of the different steps of the central dogma can be integrated to reveal its intricate temporal dependencies.


MATERIALS AND METHODS

All animal procedures were conducted in accordance with institutional guidelines for the care and use of laboratory animals as approved by the Institutional Animal Care and Use Committee of the University of Kentucky. For initial time course experiments, male and female C57BL/6J mice 4–6 mo of age (Jackson Laboratory, Bar Harbor, ME) were housed in a temperature- and humidity-controlled room and maintained on a 14:10 h light:dark cycle with food and water ad libitum. For myonuclear accretion experiments, male and female Pax7-DTA mice were used as previously described (McCarthy et al., 2011 Fry et al., 2014 Lee et al., 2015). Briefly, these mice allow for the inducible and specific depletion of the primary stem cell (satellite cell) in adult skeletal muscle after administration of tamoxifen. At 4 mo of age, Pax7-DTA mice were administered by intraperitoneal injection either vehicle (15% ethanol in sunflower seed oil) or tamoxifen (2 mg/d) for 5 consecutive days, followed by a 2-wk washout period before synergist ablation surgery.

The mice were subjected to bilateral synergist ablation surgery to induce hypertrophy of the plantaris muscle as previously described in detail (McCarthy and Esser, 2007). Briefly, after anesthetization with a mixture of 95% oxygen and 5% isoflurane gas, the soleus and the majority of the gastrocnemius muscles were surgically excised, with particular attention made to ensure that the neural and vascular supply remained intact and undamaged for the remaining plantaris muscle. Sham surgery controls involved similar procedures without gastrocnemius and soleus muscle excision. After recovery from surgery, mice were anesthetized at the designated time point by an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), and plantaris muscles were excised and weighed. Tissue used for RNA was flash frozen in liquid nitrogen and stored at −80°C until further use. Tissue used for microscopy was pinned to a cork block at resting length, covered with a thin layer of Tissue Tek optimum cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA), and then quickly frozen in liquid nitrogen–cooled isopentane and stored at −80°C until sectioning. For time-course experiments, plantaris muscle was collected at 3, 7, and 14 d after the surgery (SA3, SA7, and SA14, respectively n = 5 per time point). Control plantaris muscle (n = 5) was collected from mice subjected to a sham synergist ablation surgery at each of the time points (Sham). For nuclear accretion experiments, plantaris was collected 14 d after surgery (n = 6). After collection of the plantaris muscle, the mice were killed by cervical dislocation under anesthesia.

Nascent RNA labeling

For nascent RNA visualization and expression experiments, mice were given an intraperitoneal injection of 2 mg of EU (Jena Biosciences, Jena, Germany) suspended in sterile phosphate-buffered saline 5 h before being killed. Five hours was chosen after it was determined this pulse period allows for maximal visual detection of nascent RNA within the nucleus in both sham and overloaded skeletal muscle (Supplemental Figure S9). EU is a uridine analogue that is incorporated specifically into newly synthesized RNA (Jao and Salic, 2008). Each EU molecule had been modified to contain an alkyne group, which can then be used for detection with molecules containing an azide group via copper-mediated “click” chemistry.

Histochemistry

Frozen tissue was sectioned (7 μm), air dried for ∼20 min, and then immediately fixed in 4% paraformaldehyde. For EU-RNA detection, sections were incubated in a solution containing Tris base (100 mM), copper(II) sulfate (4 mM), biotin-conjugated azide (100 μm Jena Biosciences), and ascorbic acid (100 mM) for 30 min at room temperature and washed, and this procedure was repeated. Sections were then incubated in Texas red–streptavidin (1:150 Vector Labs, Burlingame, CA) for 60 min at room temperature. Sections were postfixed in 4% paraformaldehyde (PFA), blocked in 1% bovine serum albumin (BSA) for 1 h, and then incubated with an anti-laminin antibody (1:100 Sigma-Aldrich, St. Louis, MO) overnight at 4°C. Sections were washed, and laminin immunoreactivity was visualized with Alexa Fluor 488 goat anti-rabbit secondary (1:500 Invitrogen, Carlsbad, CA) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI). For histine 3 lysine 9 acetylation (H3K9ac), sections were air dried for 1 h, fixed in 4% PFA, blocked using 1% BSA, and incubated in primary antibody (1:1000 ab10812 Abcam, Cambridge, MA) overnight 4°C using a 1:500 dilution in 1% BSA. H3K9ac was visualized using Alexa Fluor 594 chicken anti-rabbit secondary (1:500 Invitrogen). Sections were washed and reacted with laminin antibody and DAPI as described.

For Pax7 detection, sections were fixed in 4% PFA, followed by epitope retrieval using sodium citrate (10 mM, pH 6.5) at 92°C for 20 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in phosphate-buffered saline for 7 min, followed by an additional blocking step with Mouse-on-Mouse Blocking Reagent (Vector Laboratories). Incubation with Pax7 antibody (1:100 Developmental Studies Hybridoma Bank, Iowa City, IA) was followed by incubation with the biotin-conjugated secondary antibody (1:1000 Jackson ImmunoResearch, West Grove, PA), and the signal was amplified using streptavidin–horseradish peroxidase included within a tyramide signal amplification kit (Invitrogen). Tyramide signal amplification–Alexa Fluor 488 or 594 was used to visualize antibody binding.

Image acquisition and quantification

Images were acquired using a Zeiss upright microscope (AxioImager M1 Carl Zeiss, Oberkochen, Germany) at 20× magnification, and analysis was carried out using the AxioVision Rel 4.8 software (Zeiss). During image acquisition of EU-RNA and H3K9ac, careful consideration was taken to select an exposure that fell within the linear portion of the dynamic range of the camera to avoid overexposure. EU-RNA– and H3K9ac-positive staining was determined by thresholding a lower-density cutoff, which was then maintained for all images analyzed. An integrated density value for each EU-RNA– and H3K9ac-positive area was calculated using the formula

Densities across different exposure times were normalized, taking into account the dark pixel intensity (Pang et al., 2012). EU-RNA– and H3K9ac-positive nuclei were considered to be from myogenic cells when a DAPI-positive nucleus fell inside the laminin stain of the myofiber or nonmyogenic when the nucleus fell outside the laminin border. Nuclei that did not contain an EU-RNA– or H3K9ac-positive area were manually counted. Based on the results of the EU-RNA experiments, H3K9ac quantification was restricted to myogenic cells. For each animal, an average of 786 ± 37 and 609 ± 48 fibers were analyzed per cross-sectional area for C57BL6/J and Pax7-DTA mouse strains, respectively.

Cell-size global transcription analysis

Myofiber cross-sectional area was quantified on the same images used for EU-RNA quantification. Cross-sectional area was determined using automated segmentation of the myofiber by selecting an intensity threshold below that of the laminin fluorescent stain. For each animal, an average of 509 ± 36 fibers per cross-sectional area were analyzed. A relative fiber frequency based on cross-sectional area was generated for each animal and averaged for each time point. Based on the inherent variability between the cross-sectional area on male and female mice, the cross-sectional area of male mice was normalized using a correction factor (0.779) derived from a large cohort of control mice (n = 24 female and n = 24 male) from previous studies (McCarthy et al., 2011 Fry et al., 2014) and unpublished data.

To determine the influence of cell size on transcriptional output, data from the cross-sectional and EU-RNA analyses were aligned only myofibers that contained an EU-RNA–positive area were included in the analysis. If a single myofiber contained multiple EU-RNA labels (i.e., multiple nuclei), the data were summed to give a total amount of EU-RNA for each fiber. About 200 myofibers were quantified per animal, with each EU-RNA quantity normalized to the geometric mean of all EU-RNA quantities. This was performed to facilitate comparisons across animals with different overall labeling intensities, since the primary objective was to determine how variation in cell size affected EU-RNA levels within each animal.

Nascent RNA affinity purification and cDNA synthesis

Total RNA was prepared from plantaris muscle using TRIzol reagent (Invitrogen) according to the manufacturer’s directions. RNA samples were treated with TURBO DNase (Ambion, Austin, TX) to remove genomic DNA contamination. Total RNA concentration and purity were assessed by measuring the optical density (230, 260, and 280 nm) with a Nanodrop 1000 Spectrophotometer (ThermoFisher Scientific, Wilmington, DE). Nascent RNA was affinity purified from 4.5 μg of total RNA using the commercially available Click-iT Nascent RNA Capture Kit (Life Technologies, Carlsbad, CA). cDNA was generated on 500 ng of total RNA and all EU affinity-purified RNA using the SuperScript VILO cDNA Synthesis Kit (Life Technologies).

Cell type–specific transcript analysis

qPCR was performed using cDNA generated from both total and nascent RNA. To minimize the effect of transcript half-life on the enrichment analysis, cell type–specific transcripts were selected based on a narrow range of half-lives (Sharova et al., 2009). Short-lived transcripts had a half-life of 4-6.5 h, whereas intermediate-lived transcripts had a half-life of 16–18 h. Short-lived, cell type–specific transcripts included muscle RING-finger protein-1 (Murf1 or Trim63 myofiber), paired box 7 (Pax7 satellite cell), epidermal growth factor-like module–containing mucin-like hormone receptor–like 1 (Emr1 or F4/80 macrophage), transcription factor 4 (Tcf4 fibroblast), and endothelial-specific receptor tyrosine kinase (Tek or Tie2 endothelial cell). Intermediate-lived (16 to 18-h half-life) cell type–specific transcripts included γ-sarcoglycan (Scgc myofiber), colony-stimulating factor 2 receptor, α, low-affinity (granulocyte-macrophage) (Csf2ra macrophage), collagen, type III, α,1 (Col3a1 fibroblast) and platelet/endothelial cell adhesion molecule 1 (CD31 or Pecam1 endothelial cell). The limited number and low expression of satellite cell–specific genes precluded detection of an intermediate-lived transcript for satellite cells. Cycle threshold (Ct) was determined using KiCqStart qPCR ReadyMix (KCQS07 Sigma-Aldrich) using 5 μl of diluted cDNA (1/20 dilution from stock cDNA mixture). qPCR was performed using an ABI 7900HT Fast RT-PCR system (Invitrogen), and Ct was determined using ABI 7900HT Sequence Detection Systems software, version 2.3. Absolute quantification was achieved by exponential conversion of the Ct using the qPCR efficiency, which was estimated from standard curves obtained by serial dilutions (one-log range) of a pooled sample for each RT set. Relative quantification for each transcript was determined using the relative standard curve method, in which absolute values are generated for both the transcript of interest and four stable transcripts (Rn7sk, Gapdh, Tmx4, and Capzb) known to show little change in abundance in the synergist ablation model of skeletal muscle hypertrophy (Chaillou et al., 2013). The geometric mean of the four stable transcripts was used to normalize the data (Vandesompele et al., 2002). Nascent RNA enrichment for each mRNA was determined by calculating its fold difference between nascent RNA and total RNA. Primers for all transcripts are listed in Supplemental Table S1.

Transcript stability assessment

Transcript stability was assessed by qPCR for myelocytomatosis oncogene (cMyc), myostatin (Mstn), F-box protein 32 (Mafbx, Fbxo32, atrogin-1), and insulin-like growth factor 1 (Igf1). Transcript stability was calculated as the ratio between the amounts of nascent RNA and total RNA (nascent RNA:total RNA). All comparisons were made relative to the sham condition ratio. A decrease in the nascent RNA:total RNA ratio indicated that less mRNA was actively being transcribed relative to the total RNA fraction, which was interpreted as an increase in transcript stability. Conversely, an increase in the nascent RNA:total RNA ratio indicated that more mRNA was actively being transcribed relative to the total RNA fraction, which was interpreted as a decrease in transcript stability.

Statistical analyses

Data were analyzed using either a one-way or two-way analysis of variance, followed by Tukey’s post hoc analysis to discriminate which means differed. In cases of nonnormal distribution, data were log transformed. For all analyses, significance was set at p < 0.05. Data are presented as mean ± SEM.



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