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I was reading a paper, http://www.pnas.org/content/109/8/E471.short, where the authors claim that (e475)
Translation of the TfR (Transferrin Receptor) is regulated through sequences in the 3' and 5' UTRs (untranslated regions) of its mRNA. To study the regulation of TfR levels independently of translational control, we measured the levels of TfR-EGFP expressed from a plasmid lacking the regulatory sequences present in the endogenous TfR mRNA (Fig. 6A). Endogenous and GFP-tagged expressed TfR levels were reduced equally in response to Rabenosyn-5 depletion, ruling out an effect of this protein on transcriptional or translational control of TfR expression.
I believe I understand that the effect can not be post-translational, since TfR-GFP has no region for translational regulation and so it cannot be regulated by any translationally active regulating factor.
My question is: Wouldn't the statement above, that Rabenosyn-5 has no effect on protein transcription, require a proof that mRNA levels are not affected by this protein?
Fig. 6A, http://www.pnas.org/content/109/8/E471.figures-only
From the Methods section:
Human TfR in plasmid cDNA was a gift from Tim McGraw (Weill-Cornell Medical College, New York, NY). Human TfR cDNA was subcloned in frame with EGFP in the Clontech pEGFP-N1 vector at the XhoI and BamHI restriction sites.
This TfR-GFP fusion protein does not have the endogenous TfR promoter. So it is not likely to be regulated by any TF that would directly regulate TfR transcription.
So they conclude that Rabenosyn-5 does not specifically regulate TfR expression.
Like transcription, translation is controlled by proteins that bind and initiate the process. In translation, the complex that assembles to start the process is referred to as the initiation complex. Regulation of the formation of this complex can increase or decrease rates of translation (Figure 1).
Figure 1 Gene expression can be controlled by factors that bind the translation initiation complex.
B. Translation Regulation
Since mRNAs are made to be translated, it is likely that by default, they are! We know that CAP and poly(A) tails on mRNAs are required for efficient translation because mRNAs engineered to lack one and/or the other are poorly translated. Also, there is little evidence to that cells modify the process of capping or polyadenylation, or the structures themselves.
Translation regulation typically targets initiation. It may be global, affecting the synthesis of many polypeptides at once, or specific, affecting a single polypeptide. Global regulation involves changes in the activity of eukaryotic initiation factors (eIFs) that would typically affect all cellular protein synthesis. Specific regulation involves binding sequences or regions on one or a few mRNAs that recognize and bind specific regulatory proteins and/or other molecules. That binding controls translation of only those mRNAs, without affecting general protein biosynthesis. mRNA structural features involved in translation and in translation regulation are illustrated below.
We will consider three examples of translational control of gene expression.
1. Specific Translation Control by mRNA Binding Proteins
Ferritin is a cellular iron-storage protein made up of heavy and light chain polypeptides. Translation of ferritin in iron-deficient cells is inhibited. In the absence of ferritin production, ferritin-iron complexes release iron for metabolic use. The 5&rsquo-UTR of mRNAs for both chains contain stem-loop binding sites that specifically recognize iron regulatory proteins (IRP1, IRP2). When ferritin mRNAs are bound to IRPs, translation initiation is blocked. The inhibition of ferritin translation by IRPs is illustrated below.
Normally, the initiation complex scans the 5&rsquo-UTR of an mRNA. When it finds the normal translation start site, it can bind the large subunit and begin translating the polypeptide. In iron-deficient cells, scanning by the initiation complex is thought to be physically blocked by steric hindrance.
2. Coordinating Heme & Globin Synthesis
Consider that reticulocytes (the precursors to erythrocytes, the red blood cells in mammals) synthesize globin proteins. They also synthesize heme, an iron-bound porphyrin-ring molecule. Each globin must bind to a single heme to make a hemoglobin protein subunit. Clearly, it would not do for a reticulocyte to make too much globin protein and not enough heme, or vice versa. It turns out that hemin (a precursor to heme) regulates the initiation of translation of both (alpha ) and (eta ) globin mRNAs. Recall that, to sustain globin mRNA translation, the GDP-eIF2 generated after each cycle of translation elongation must be exchanged for fresh GTP. This is facilitated by the eIF2B initiation factor. eIF2B can exist in phosphorylated (inactive) or un-phosphorylated (active) states. Making sure that globin is not under- or overproduced relative to heme biosynthesis involves controlling levels of active vs. inactive eIF2B by hemin. Hemin accumulates when there is not enough globin polypeptide to combine with heme in the cell. Excess hemin binds and inactivates an HCR kinase, preventing phosphorylation of eIF2B. Since unphosphorylated eIF2B is active, it facilitates the GTP/GDP swap needed to allow continued translation. Thus, ongoing initiation ensures that globin mRNA translation can keep up with heme levels. In other words, if hemin production gets ahead of globin, it will promote more globin translation.
When globin and heme levels become approximately equimolar, hemin is no longer in excess. It then dissociates from the active HCR kinase. The now- active kinase catalyzes eIF2B phosphorylation. Phospho-eIF2B is inactive, and cannot facilitate the GTP/GDP swap on eIF2. Globin mRNA translation initiation, thus blocked, allows a lower rate of globin polypeptide translation to keep pace with heme synthesis. The regulation of globin mRNA translation initiation by hemin is shown below.
3. Translational Regulation of Yeast GCN4
Like the coordination of heme and globin production, the regulation of the GCN4 protein is based on controlling the ability of the cells to swap GTP for GDP on eIF2. However, this regulation is quite a bit more complex, despite the fact that yeast is a more primitive eukaryote! GCN4 is a global transcription factor that controls the transcription of as many as 30 genes in pathways for the synthesis of 19 out of the 20 amino acids! The discovery that amino acid starvation caused yeast cells to increase their production of amino acids in the cells led to the discovery the General Amino Acid Control (GAAC) mechanism involving GCN4. GCN is short for General Control Nondepressible, referring to its global, positive regulatory effects. It turns out that the GCN4 protein is also involved in stress gene expression, glycogen homeostasis, purine biosynthesis&hellip, in fact in the action of up to 10% of all yeast genes! Here we focus on the GAAC mechanism.
Yeast cells provided with ample amino acids do not need to synthesize them. Under these conditions, GCN4 is present at basal (i.e., low) levels. When the cells are starved of amino acids, GCN4 levels increase as much as ten-fold within two hours, resulting in an increase in general amino acid synthesis. This rapid response occurs because amino acid starvation signals an increase in the activity of GCN2, a protein kinase. The GCN2 kinase catalyzes phosphorylation of GDPeIF2. As we have already seen, phosphorylated eIF2B cannot exchange GTP for GDP on the eIF2, in this case with the results shown below.
There is a paradox here. You would expect a slowdown in GTP-eIF2 regeneration to inhibit overall protein synthesis, and it does. However, the reduced levels of GTP-eIF2 somehow also stimulate translation of the GCN4 mRNA, leading to increased transcription of the amino acid synthesis genes. In other words, amino acid starvation leads yeast cells to use available substrates to make their own amino acids in order that protein synthesis can continue&hellip at the same time as initiation complex formation is disabled!
Let&rsquos accept that paradox for now, and look at how amino acid starvation leads to increased translation of the GCN4 protein and the up-regulation of amino acid biosynthesis pathways. To begin with, we are going to need to understand the structure of GCN4 mRNA. In the illustration below, note the 4 short uORFs in the 5&rsquoUTR of the RNA these play a key role in GCN4 translation regulation.
We noted earlier that when a Ternary Complex (TC)-associated 40S ribosomal subunit scans an mRNAs and find the ORF start sites for its polypeptide, initiation complexes form, 60S ribosomal subunits bind and translation starts. GCN4 mRNA has four uORFs in its 5&rsquo UTR. While uORFs encode only a few amino acids before encountering a stop codon, they can also be recognized during scanning. When TCs and 40S subunits are plentiful, they seem to engage uORFs in preference to the GCN4 coding region ORF, as illustrated below.
Under these conditions, active eIF2B allows the GTP/GDP swap on GDP-eIF2, leading to efficient GTP-eIF2 recycling and high TC levels. TCs bind small subunits during scanning and/or at the start sites of uORFs, forming initiation complexes that then bind 60S ribosomal subunits and begin uORF translation. The effect is to slow down scanning past the uORFs, thereby inhibiting initiation complex formation at the actual GCN4 ORF.
What happens in amino acid-starved cultures of yeast cells, when GTP-eIF2 cannot be efficiently regenerated and TCs are in short supply? To review, amino acid starvation signals an increase in GCN2 kinase activity resulting in phosphorylation and inactivation of eIF2B. Inactive phospho-eIF2 will not facilitate the GTP/GDP swap at GDP-eIF2, inhibiting overall protein synthesis. The resulting reduction in GTP-eIF2 also lowers the levels of TC and TC-associated 40S subunits. The illustration below shows how this phenomenon up-regulates GCN4 translation, even as the translation of other mRNAs has declined.
Regulation of Gene Expression: Negative and Positive Regulation
The two types of gene expression regulation are: (1) Negative Regulation and (2) Positive Regulation. And also discuss about some important terms used in connection with the regulation of gene expression.
Most of the genes of an organism produce specific proteins (enzymes), which, in turn produce specific phenotypes. The genes whose mRNA transcripts are translated into protein are known as structural genes. Every cell of an organism possesses all the structural genes normally present in the species, but only a small fraction of them are functional in any cell at a given time.
In prokaryotes, cells generally synthesize only those enzymes which they need in a given environment. For example, E. coli cells grown in the presence of lactose produce abundant (up to 3000 molecules/cell) β-galactosidase, the enzyme that hydrolyses lactose. However, very little of this enzyme (less than 3 molecules/cell) is produced in the absence of lactose.
In eukaryotes, the cells of different organs produce different proteins needed for their function. Red blood cells contain a high concentration of hemoglobin, while leucocytes (white blood cells) have no hemoglobin at all.
Apparently, there is a precise control on the kinds of proteins or enzymes product in a given tissue or cell at a given time. Such a control on gene activity, i.e., protein production, that permits the function of only those genes whose products are required in a given cell at a given time is termed as gene regulation.
Synthesis of enzyme depends mainly on two factors in a degradative process, the synthesis of enzyme depends on the availability of the molecule to be degraded. If the molecule is in more quantity, the enzyme synthesis will be more and vice versa. In a biosynthetic pathway, the synthesis of an enzyme is controlled by the end product. If the end product is more, the enzyme synthesis will be less and vice versa.
There are two types of gene regulation, viz:
(1) Negative regulation, and
(1) In negative regulation:
An inhibitor is present in the cell/system, that prevents transcription by inactivating the promoter. This inhibitor is known as repressor. For initiation of transcription, an inducer is required. Inducer acts as antagonist of the repressor. In the negative regulation, absence of product increases the enzyme synthesis and presence of the product decreases the synthesis.
(2) In positive regulation:
An effector molecule (which may be a protein or a molecular complex) activates the promoter for transcription. In a degradative system, either negative or positive mechanism may operate, while in a biosynthetic pathway negative mechanism operates (e.g., lac operon).
The phenomenon of gene expression can be elaborated further such as given below:
1. Gene expression is the mechanism at the molecular level by which a gene is able to express itself in the phenotype of an organism.
2. The mechanism of gene expression involves biochemical genetics. It consists of synthesis of specific RNAs, polypeptides, structural proteins, proteinaceous bio-chemicals or enzymes which control the structure or functioning of specific traits.
3. Gene regulation is the mechanism of switching off and switching on of the genes depending upon the requirement of the cells and the state of development.
4. It is because of this regulation that certain proteins are synthesized in as few as 5-10 molecules while others are formed in more than 100,000 molecules per cell.
5. There are two types of gene regulations positive and negative.
6. In negative gene regulation the genes continue expressing their effect till their activity is suppressed.
7. This type of gene regulation is also called repressible regulation.
8. The repression is due to a product of regulatory genes.
9. Positive gene regulation is the one in which the genes remain non-expressed unless and until they are induced to do it.
10. It is, therefore called inducible regulation.
11. Here a product removes d biochemical that keeps the genes in non-expressed state.
12. As the genes express their effect through enzymes, their enzymes are also called inducible enzymes and repressible enzymes.
Gene regulation is exerted at four levels:
1. Transcriptional level when primary transcript is formed.
2. Processing level when splicing and terminal additions are made.
3. Transport of mRNA out of nucleus into cytoplasm.
Important Terms used in Connection with the Regulation of Gene Expression:
In operon, protein molecules which prevent transcription. The process of inhibition of transcription is called repression.
The substance that allows initiation of transcription (e.g., lactose in lac operon). Such process is known as induction.
A combination of repressor and a metabolite which prevents protein synthesis. Such process is known as co-repression.
An enzyme whose production is enhanced by adding the substrate in the culture medium. Such system is called inducible system.
An enzyme whose production can be inhibited by adding an end product. Such system is known as repressible system.
6. Constitutive Enzyme:
An enzyme whose production is constant irrespective of metabolic state of the cell.
Inhibition of transcription by repressor through inactivation of promoter, e.g., in lac operon.
Enhancement of transcription by an effector molecule through activation of pro-motor.
DNA Modification: Methylation
The DNA molecule itself can also be modified by methylation. DNA methylation occurs within very specific regions called CpG islands. These are regions of the DNA that contain a high frequency of cytosine and guanine dinucleotide DNA pairs (CG). The ‘p’ stands for the phosphate group that is part of the phosphodiester bond between the cytosine and guanine (CG). Figure 5-4 illustrates the difference between a gene containing CpG island and a normal gene promoter sequence. These are found in the promoter regions of genes. The cytosine base of the CG pair can be methylated, which often causes the gene to be silenced because transcription factors can no longer bind to the promoter, as the methyl group blocks that interaction. In some cases, genes that are silenced during the development of the gametes of one parent are transmitted in their silenced condition to the offspring. Such genes are said to be imprinted. Parental diet or other environmental conditions can affect the methylation patterns of genes, which in turn modifies gene expression. Additionally, external environmental changes after the offspring are born can dynamically alter the methylation status of genes, which can impact human health (Figure 5-4). Some such stimuli are trauma, physical activity, smoking, alcohol consumption, environmental pollutants, and obesity.
The methylation of histones can influence the methylation of DNA. DNA methyltransferases (enzymes important in methylating DNA) appear to be attracted to chromatin regions with specific histone modifications. Highly methylated (hypermethylated) DNA regions with deacetylated (lacking acetyl groups) histones are tightly coiled and transcriptionally inactive.
Figure 5-4: Example of DNA sequence that is a known CpG island. The CG sequence is in yellow. Notice the extensive amount of CG dinucleotides. The ATG in red, this denotes the start of translation for this gene. This signifies that this region is likely to be highly methylated, silencing transcription of this gene. Alterations in the sequence of CpG islands can result in misregulation of genes and inappropriate production of proteins that ultimately can result in disease phenotypes. The image on the right represents an area of DNA that is not regulated by CpG islands.
Epigenetic changes are not permanent in the same way a mutation in the DNA sequence can be, although they often persist through multiple rounds of cell division and may be heritable.
Chromatin remodeling alters the chromosomal structure (open or closed) as needed. If a gene is to be transcribed, the histone proteins and DNA in the chromosomal region encoding that gene are modified in a way that opens the promoter region to allow RNA polymerase and other proteins, called transcription factors, to bind and initiate transcription. If a gene is to remain turned off, or silenced, the histone proteins and DNA have different modifications that signal a closed chromosomal configuration. In this closed configuration, the RNA polymerase and transcription factors do not have access to the DNA, and transcription cannot occur ( Figure 5-5).
Figure 5-5: Histone proteins and DNA nucleotides can be modified chemically. Modifications affect nucleosome spacing and gene expression. (credit: modification of work by NIH)
So how does a plant know when to senescence?
During the workshop it became clear that plant senescence is regulated at several levels. However, trying to separate the causes and consequences of senescence raises several ‘chicken and egg’ problems. For example, what comes first – transcriptional or post-transcriptional regulation, age or stress, metabolites or gene expression? Latest research makes it possible to suggest a model in which age-dependent changes to chromatin and possibly also telomere structure could make a leaf or a plant competent to react to senescence-inducing environmental stimuli, such as day length or stress (Fig. 1). These stimuli could regulate gene expression either directly or, for example, via plant hormones, metabolites or reactive oxygen species. Changes in metabolism are likely to be the cause as well as the consequence of transcriptional and post-transcriptional changes. Furthermore, a possibly age-related switch in post-translational modification, such as hypusination of eIF5A, could regulate protein synthesis. While downstream processes, such as the degradation of chlorophyll, are highly conserved ( Armstead et al., 2007 ), there are many different ways in which senescence can be induced. Unravelling the causal relationships in the regulatory network therefore remains a challenging task.
Hypothetical model for regulatory mechanisms that control senescence in plants.
16.11 Critical Thinking Questions
All the cells of one organisms share the genome. However, during development, some cells develop into skin cells while others develop into muscle cells. How can the same genetic instructions result in two different cell types in the same organism? Thoroughly explain your answer.
- When lactose and glucose are present in the medium, transcription of the lac operon is induced.
- When lactose is present but glucose is absent, the lac operon is repressed.
- Lactose acts as an inducer of the lac operon when glucose is absent.
- Lactose acts as an inducer of the lac operon when glucose is present.
- Mutation in structural genes will stop transcription.
- Mutated lacY will produce an abnormal β galactosidase protein.
- Mutated lacA will produce a protein that will transfer an acetyl group to β galactosidase.
- Transcription will continue but lactose will not be metabolized properly.
In some diseases, alteration to epigenetic modifications turns off genes that are normally expressed. Hypothetically, how could you reverse this process to turn these genes back on?
- In new seedlings, histone acetylations are present upon cold exposure, methylation occurs.
- In new seedlings, histone deacetylations are present upon cold exposure, methylation occurs.
- In new seedlings, histone methylations are present upon cold exposure, acetylation occurs.
- In new seedlings, histone methylations are present upon cold exposure, deacetylation occurs.
- Mutated promoters decrease the rate of transcription by altering the binding site for the transcription factor.
- Mutated promoters increase the rate of transcription by altering the binding site for the transcription factor.
- Mutated promoters alter the binding site for transcription factors to increase or decrease the rate of transcription.
- Mutated promoters alter the binding site for transcription factors and thereby cease transcription of the adjacent gene.
- The transcription rate would increase, altering cell function.
- The transcription rate would decrease, inhibiting cell functions.
- The transcription rate decreases due to clogging of the transcription factors.
- The transcription rate increases due to clogging of the transcription factors.
The wnt transcription pathway is responsible for key changes during animal development. Based on the transcription pathway shown below. In this diagram, arrows indicate the transformation of one substance into another. Square lines, or the lines with no arrowheads, indicate inhibition of the product below the line. Based on this, how would increased wnt gene expression affect the expression of Bar-1?
- RBPs can bind first to the RNA, thus preventing the binding of miRNA, which degrades RNA.
- RBPs bind the miRNA, thereby protecting the mRNA from degradation.
- RBPs methylate miRNA to inhibit its function and thus stop mRNA degradation.
- RBPs direct miRNA degradation with the help of a DICER protein complex.
- UV rays can alter methylation and acetylation of proteins.
- RNA binding proteins are modified through phosphorylation.
- External stimuli can cause deacetylation and demethylation of the transcript.
- UV rays can cause dimerization of the RNA binding proteins.
- Phosphorylation of proteins can alter translation, RNA shuttling, RNA stability or post transcriptional modification.
- Phosphorylation of proteins can alter DNA replication, cell division, pathogen recognition and RNA stability.
- Phosphorylated proteins affect only translation and can cause cancer by altering the p53 function.
- Phosphorylated proteins affect only RNA shuttling, RNA stability, and post-translational modifications.
- UV rays could cause methylation and deacetylation of the genes that could alter the accessibility and transcription of DNA.
- The UV rays could cause phosphorylation and acetylation of the DNA and histones which could alter the transcriptional capabilities of the DNA.
- UV rays could cause methylation and phosphorylation of the DNA bases which could become dimerized rendering no accessibility of DNA.
- The UV rays can cause methylation and acetylation of histones making the DNA more tightly packed and leading to inaccessibility.
- These drugs maintain the demethylated and the acetylated forms of the DNA to keep transcription of necessary genes “on”.
- The demethylated and the acetylated forms of the DNA are reversed when the silenced gene is expressed.
- The drug methylates and acetylates the silenced genes to turn them back “on”.
- Drugs maintain DNA methylation and acetylation to silence unimportant genes in cancer cells.
- Understanding gene expression patterns in cancer cells will identify the faulty genes, which is helpful in providing the relevant drug treatment.
- Understanding gene expression will help diagnose tumor cells for antigen therapy.
- Gene profiling would identify the target genes of the cancer-causing pathogens.
- Breast cancer patients who do not express EGFR can respond to anti-EGFR therapy.
- Personalized medicines would vary based on the type of mutations and the gene’s expression pattern.
- The medicines are given based on the type of tumor found in the body of an individual.
- The personalized medicines are provided based only on the symptoms of the patient.
- The medicines tend to vary depending on the severity and the stage of the cancer.
- You are here:
- Chapter 16 Gene Regulation
- 16.11 Critical Thinking Questions
This text is based on Openstax Biology for AP Courses, Senior Contributing Authors Julianne Zedalis, The Bishop's School in La Jolla, CA, John Eggebrecht, Cornell University Contributing Authors Yael Avissar, Rhode Island College, Jung Choi, Georgia Institute of Technology, Jean DeSaix, University of North Carolina at Chapel Hill, Vladimir Jurukovski, Suffolk County Community College, Connie Rye, East Mississippi Community College, Robert Wise, University of Wisconsin, Oshkosh
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 Unported License, with no additional restrictions
How is Gene Expression Regulated in Prokaryotes and Eukaryotes
Gene expression is the process of transcription of DNA into RNA, followed by translation into proteins. It is a tightly regulated process in both prokaryotes and eukaryotes. The regulation of gene expression is involved in the production of either increased or decreased amount of gene products. A wide range of mechanisms is involved in the regulation of gene expression.
- Replication level – Mutations may cause alterations of the gene expression.
- Transcriptional level – Activators and repressors regulate the transcription.
- Post-transcriptional level – RNA splicing is involved in the regulation of gene expression at post-transcriptional level.
- Translational level – The translation of a mRNA molecule into an amino acid sequence of a protein is controlled by various processes such as RNA interference pathway.
- Post-translational level – The synthesis of a protein can be regulated by controlling the post-translational modifications.
Different levels of regulation of gene expression are shown in figure 1.
Figure 1: Regulation of Gene Expression
Glossary of techniques for studying protein synthesis and translation regulation
Several different techniques have been employed to study translation regulation, from the old (but still used) pulse-chase labeling to the more modern techniques that take advantage of the whole-genome sequencing methods. Here several of the techniques are explained and some protocols cited.
Pulse labeling is a technique employed for identifying the rate of protein synthesis per unit of time. The method is based on the incorporation by the plant of radioactively labeled amino acids, usually [35 S ]methionine/cysteine, over a series of time points. Proteins are then extracted and the newly synthesized proteins that have incorporated the labeled amino acid are detected by SDS-PAGE followed by autoradiography and densitometry or by scintillation counting. Protocols that avoid the use of radioactive labeling and employ unnatural amino acids instead have also been developed (Schmidt et al., 2009 Wang et al., 2017a ), but have not yet been implemented in plants.
Cell-free translation systems are useful for studying the molecular mechanisms of translation in vitro. The most widely utilized cell-free system employed in the studies of plant translation is wheat germ extract (Olliver et al., 1996 ), but a cell-free Arabidopsis system has also become available (Murota et al., 2011 ).
Polysome fractionation is a classical technique that allows for the study of the translational state of a whole plant or a specific tissue, i.e. to infer the translational efficiency at the global level or of a particular mRNA of interest. This method is still widely practiced and involves the use of sucrose gradients and ultracentrifugation to separate the polysomes based on the number of ribosomes that they contain. The centrifuged sample resolved in the gradient is then split into fractions (with the lower denser fractions containing heavier polysomes and upper lighter fractions harboring individual ribosomal subunits) and the RNAs are isolated from the fractions to proceed to expression studies, such as Northern blot, RT-PCR, microarray or next-generation sequencing (Mustroph et al., 2009b ).
Translating ribosome affinity purification (TRAP) is an affinity-chromatography-based ribosome purification method in which a specific ribosomal protein is tagged and then immunoprecipitated to isolate the mRNAs bound by the tagged ribosomes. Next-generation sequencing of the ribosome-bound mRNAs provides information about the translational state of the cell (Zanetti et al., 2005 Mustroph et al., 2009b ). This technique, like polysome fractionation, relies on polysomal RNA as a proxy for translation, but cannot distinguish between translating ribosomes and those stalled on mRNAs non-productively.
Ribo-seq (aka ribosome footprinting) is a higher-resolution technology that reveals the exact position of the ribosomes on an mRNA and allows discrimination between ribosomes stalled in 5′ UTRs and those positioned in coding regions. Polysomal RNAs in this case are isolated either using ultracentrifugation through a sucrose cushion or using column-based gel filtration methods and then treated with an RNase. As each translating ribosome shields a short stretch on its respective mRNA, with the exposed parts of all transcripts being degraded by the RNase treatment, sequencing of the surviving footprints using next-generation approaches provides codon-level information on where the ribosomes were on each and every transcript. Initially developed for yeast (Ingolia et al., 2009 ), efficient Ribo-seq protocols have also been implemented in plants (Hsu et al., 2016 Merchante et al., 2016 ).
Sequencing-based structure mapping of RNA secondary structure with double-strand- and single-strand-specific nucleases is a technique that allows for a genome-wide analysis of RNA secondary structure by mapping double-stranded (ds) and single-stranded (ss) regions in RNA molecules inferred from the digestion of RNAs with ds- and ss-specific nucleases. Complex RNA samples are split into two and subjected to alternative nuclease treatments. ssRNAs are degraded by the use of ssRNases and dsRNAs are purified and converted into sequencing libraries. In parallel, the second half of the sample is treated with dsRNases, and the remaining ssRNAs are isolated and converted into sequencing libraries. Comparative analysis of the reads allows the generation of an unbiased and comprehensive collection of RNA secondary structure models (Li et al., 2012b ).
Structure-seq is a state-of-the-art technique that combines dimethyl sulfate (DMS) methylation with next-generation sequencing to quantitatively measure RNA secondary structure at a genome-wide level and single-nucleotide resolution, both in vivo and in vitro. DMS methylates unpaired As and Cs (all paired nucleotides are protected) and this results in the termination of reverse transcription products. Comparison of the sequencing results between DMS-treated and untreated samples pinpoints unpaired As and Cs. Thus, high DMS reactivity indicates less structured regions which account for loops, bulges, mismatches or joining regions (Ding et al., 2014 ).
In addition, new techniques that study mRNA degradation intermediates at a genome-wide level, such as parallel analysis of RNA ends (PARE) (Zhai et al., 2014 ) or genome-wide mapping of uncapped and cleaved transcripts (GMUCT) (Gregory et al., 2008 ), have been developed and aid in the study of ribosome positioning, translation and co-translational degradation. These techniques involve the capture of the 5′ monophosphorylated ends of cleaved 3′ end mRNA products and make sequencing libraries by ligating RNA adapters.