We are searching data for your request:
Upon completion, a link will appear to access the found materials.
Source: BiochemFFA_7_5.pdf. The entire textbook is available for free from the authors at http://biochem.science.oregonstate.edu/content/biochemistry-free-and-easy
So far, we have looked at the mechanism by which the information in genes (DNA) is transcribed into RNA. The newly made RNA, also known as the primary transcript is further processed before it is functional. Both prokaryotes and eukaryotes process their ribosomal and transfer RNAs. The major difference in RNA processing, however, between prokaryotes and eukaryotes, is in the processing of messenger RNAs. We will focus on the processing of mRNAs in this section. You will recall that in bacterial cells, the mRNA is translated directly as it comes off the DNA template. In eukaryotic cells, RNA synthesis, which occurs in the nucleus, is separated from the protein synthesis machinery, which is in the cytoplasm. The initial product of transcription of an mRNA is sometimes referred to as the pre-mRNA. After it has been processed and is ready to be exported from the nucleus, it is called the mature mRNA. The three main processing steps for mRNAs are (Figure 7.67):
• Capping at the 5' end
• Splicing to remove introns
• Addition of a polyA tail at the 3' end.
Although this description suggests that these processing steps occur post-transcriptionally, after the entire gene has been transcribed, there is evidence that processing occurs co-transcriptionally. That is, the steps of processing are occurring as the mRNA is being made. Proteins involved in mRNA processing have been shown to be associated with the phosphorylated C-terminal domain (CTD) of RNA polymerase II.
As might be expected, the addition of an mRNA cap at the 5’ end is the first step in mRNA processing, since the 5’end of the RNA is the first to be made. Capping occurs once the first 20-30 nucleotides of the RNA have been synthesized. The addition of the cap involves removal of a phosphate from the first nucleotide in the RNA to generate a diphosphate. This is then joined to a guanosine monophosphate which is subsequently methylated at N7 of the guanine to form the 7mG cap structure (Figure 7.68). This cap is recognized and bound by a complex of proteins that remain associated with the cap till the mRNA has been transported into the cytoplasm. The cap protects the 5' end of the mRNA from degradation by nucleases and also helps to position the mRNA correctly on the ribosomes during protein synthesis.
Eukaryotic genes have introns, noncoding regions that interrupt the gene. The mRNA copied from genes containing introns will also therefore have noncoding regions that interrupt the information in the gene. These noncoding regions must be removed (Figure 7.69) before the mRNA is sent out of the nucleus to be used to direct protein synthesis.
Introns are removed from the pre-mRNA by the activity of a complex called the spliceosome. The spliceosome is made up of proteins and small RNAs that are associated to form protein-RNA enzymes called small nuclear ribonucleoproteins or snRNPs (pronounced snurps).
The splicing machinery must be able to recognize splice junctions (i.e., where each exon ends and its associated intron begins) in order to correctly cut out the introns and join the exons to make the mature, spliced mRNA. What signals indicate exon-intron boundaries? The junctions between exons and introns are indicated by specific base sequences. The consensus sequence at the 5’ exon-intron junction (also called the 5’ splice site) is AGGURAGU. In this sequence, the intron starts with the second G (R stands for any purine). The 3' splice junction has the consensus sequence YAGRNNN, where YAG is within the intron, and RNNN is part of the exon (Y stands for any pyrimidine, and N for any nucleotide).
There is also a third important sequence within the intron, about a hundred nucleotides from the 3’ splice site, called a branch point or branch site, that is important for splicing. This site is defined by the presence of an A followed by a string of pyrimidines. The importance of this site will be seen when we consider the steps of splicing.
There are two main steps in splicing. The first step is the nucleophilic attack by the 2’OH of the branch point A on the 5' splice site (the junction of the 5' exon and the intron). As a result of a trans-esterification reaction, the 5' exon is released, and a lariat-shaped molecule composed of the 3’ exon and the intron sequence is generated (Figure 7.70). In the second step, the 3' OH of the 5’ exon attacks the 3’ splice site, and the two exons are joined together, and the lariat-shaped intron is released .
As mentioned earlier, splicing is carried out by a complex consisting of small RNAs and proteins. The five small RNAs crucial to this complex, U1, U2, U4, U5 and U6 are found associated with proteins, as snRNPs. These and many other proteins work together to facilitate splicing. Although many details remain to be worked out, it appears that components of the splicing machinery associate with the CTD of the RNA polymerase and that this association is important for efficient splicing. The assembly of the spliceosome requires the stepwise interaction of the various snRNPs and other splicing factors (Figure 7.71). The initial step in this process is the interaction of the U1 snRNP with the 5’ splice site. Additional proteins such as U2AF (AF = associated factor) are also loaded onto the pre-mRNA near the branch site. This is followed by the binding of the U2 snRNA to the branch site.
Next, a complex of the U4/U6 and U5 snRNPs is recruited to the spliceosome to generate a pre-catalytic complex. This complex undergoes rearrangements that alter RNA-RNA and protein-RNA interactions, resulting in displacement of the U4 and U1 snRNPs and the formation of the catalytically active spliceosome. This complex then carries out the two splicing steps described earlier.
On average, human genes have about 9 exons each. However, the mature mRNAs from a gene containing nine exons may not include all of them. This is because the exons in a pre-mRNA can be spliced together in different combinations to generate different mature mRNAs. This is called alternative splicing, and allows the production of many different proteins using relatively few genes, since a single RNA with many exons can, by combining different exons during splicing, create many different protein coding messages. Because of alternative splicing, each gene in our DNA gives rise, on average, to three different proteins. Alternative splicing allows the information in a single gene to be used to specify different proteins in different cell types or at different developmental stages (Figure 7.72).
The 3' end of a processed eukaryotic mRNA typically has a “poly(A) tail” consisting of about 200 adenine-containing nucleotides. These residues are added by a template-independent enzyme, poly(A)polymerase, following cleavage of the RNA at a site near the 3’ end of the new transcript. Components of the polyadenylation machinery have been shown to be associated with the CTD of the RNA polymerase, showing that all three steps of pre-mRNA processing are tightly linked to transcription. There is evidence that the polyA tail plays a role in efficient translation of the mRNA, as well as in the stability of the mRNA. Like alternative splice sites, genes can have alternative polyA sites as well (Figure 7.73).
The cap and the polyA tail on an mRNA are also indications that the mRNA is complete (i.e., not defective). Once protein-coding messages have been processed by capping, splicing and addition of a poly A tail, they are transported out of the nucleus to be translated in the cytoplasm. Mature mRNAs are sent into the cytoplasm bound to export proteins that interact with the nuclear pore complexes in the nuclear envelope (Figure 7.74). Once the mature mRNA has been translocated to the cytoplasm, it is ready to be translated.
In addition to undergoing the three processing steps outlined above, many RNAs undergo further modification called RNA editing. Editing has been observed in not only mRNAs but also in transfer RNAs and ribosomal RNAs. As the name suggests, RNA editing is a process during which the sequence of the transcript is altered post-transcriptionally. A well-studied example of RNA editing is the alteration of the sequence of the mRNA for apolipoprotein B (see also HERE). The editing results in the deamination of a cytosine in the transcript to form a uracil, at a specific location in the mRNA. This change converts the codon at this position, CAA, which encodes a glutamine, into UAA, a stop codon. The consequence of this is that a shorter version of the protein is made, when the edited transcript is translated. It is interesting that the editing of this transcript occurs in intestinal cells but not in liver cells. Thus, the protein product of the apolipoprotein B gene is longer in the liver than it is in the intestine.
Another kind of RNA editing involves the insertion or deletion of one or more nucleotides. One example of this sort of editing is seen in the mitochondrial RNAs of trypanosomes. Small guide RNAs indicate the sites at which nucleotides are inserted or deleted to produce the mRNA that is eventually translated (Figure 7.75).
The effect of either of these kinds of editing on the mRNA is that the encoded protein product is different, providing another point at which the product of expression of a gene can be controlled.
TRNA synthesis & processing
tRNAs are synthesized by RNA polymerase III, which makes precursor molecules called pre-tRNA that then undergo processing to generate mature tRNAs. The initial transcripts contain additional RNA sequences at both the 5’ and 3’ ends. Some pre-tRNAs also contain introns. These additional sequences are removed from the transcript during processing.
The 5’ leader sequence of the pre-tRNA (the additional nucleotides at the 5’-end) is removed by an unusual endonuclease called ribonuclease P (RNase P - Figure 7.76). RNase is a ribonucleoprotein complex composed of a catalytic RNA and numerous proteins. The 3’ trailer sequence (extra nucleotides at the 3’ end of the pre-tRNA) is later removed by different nucleases. All tRNAs must have a 3’ CCA sequence that is necessary for the charging of the tRNAs with amino acids. In bacteria, this CCA sequence is encoded in the tRNA gene, but in eukaryotes, the CCA sequence is added post-transcriptionally by an enzyme called tRNA nucleotidyl transferase (tRNT).
As mentioned earlier, some tRNA precursors contain an intron located in the anticodon arm. In eukaryotes, this intron is typically found immediately 3’ to the anticodon. The introns is spliced out with the help of a tRNA splicing endonuclease and a ligase.
Mature tRNAs contain a high proportion of bases other than the usual adenine (A), guanine (G), cytidine (C) and uracil (U). These unusual bases are produced by modifying the bases in the tRNA to form variants, such as pseudouridine (Figure 7.77) or dihyrouridine. Modifications to the bases are introduced into the tRNA at the final processing step by a variety of specialized enzymes. Different tRNAs have different subsets of modifications at specific locations, often the first base of the anti-codon (the wobble position).
RRNA synthesis and processing
Cells contain many copies of rRNA genes (between 100 and 2000 copies are seen in mammalian cells). These genes are organized in transcription units separated by non-transcribed spacers. Each transcription unit contains sequences coding for 18S, 5.8S and 28S rRNA, and is transcribed by RNA polymerase I into a single long transcript (47S). The 5S rRNA is separately transcribed. The sizes of ribosomal RNAs are, by convention, indicated by their sedimentation coefficients, which is a measure of their rate of sedimentation during centrifugation. Sedimentation is expressed in Svedberg units (hence the S at the end of the number) with larger numbers indicating greater mass.
The initial transcript contains 5’ and 3’ external transcribed spacers (ETS) as well as internal transcribed sequences (ITS). The primary transcript is first trimmed at both ends by nucleases to give a 45S pre-rRNA. Further processing of the pre-rRNA through cleavages guided by RNA-protein complexes containing snoRNAs (small nucleolar RNAs), gives rise to the mature 18S, 5.8S and 28S rRNAs (Figure 7.79). Ribosomal RNAs are also modified both on the ribose sugars and on the bases. Interestingly, methylation of ribose sugars is the major modification in rRNA. The modified base pseudouridine is also common in rRNA. Other modifications include base methylation, and acetylation. These modifications are thought to be important in modulating ribosome function.
Information Processing: RNA Processing
HERE & HERE
Figure 7.68 - 5’ capping of eukaryotic mRNAs
Figure 7.67 - Steps in processing of pre-mRNA
Figure 7.69 - Removal of introns from the primary transcript
Figure 7.70 - Splicing of introns
Figure 7.71 - Assembly of the spliceosome complex
HERE & HERE
Figure 7.72 - Alternative splicing leads to different forms of a protein from the same gene sequence
Figure 7.73 - Alternative poly-adenylation sites for a gene
Figure 7.74 - Structure of a mature eukaryotic mRNA
Figure 7.76 - Structure of the RNA component of ribonuclease P
Figure 7.75 - Template guided - one mechanism of RNA editing
Figure 7.78 - Sequence of a mature tRNA
Figure 7.77 - Synthesis of pseudouridine from uridine
Figure 7.79 - Processing of ribosomal RNA
HERE & HERE
Graphic images in this book were products of the work of several talented students. Links to their Web pages are below
Click HERE for
Click HERE for
Click HERE for
Click HERE for
Problem set related to this section HERE
Point by Point summary of this section HERE
To get a certificate for mastering this section of the book, click HERE
Kevin Ahern’s free iTunes U Courses - Basic / Med School / Advanced
Biochemistry Free & Easy (our other book) HERE / Facebook Page
Kevin and Indira’s Guide to Getting into Medical School - iTunes U Course / Book
To see Kevin Ahern’s OSU ecampus courses - BB 350 / BB 450 / BB 451
To register for Kevin Ahern’s OSU ecampus courses - BB 350 / BB 450 / BB 451
Biochemistry Free For All Facebook Page (please like us)
Kevin Ahern’s Web Page / Facebook Page / Taralyn Tan’s Web Page
Kevin Ahern’s free downloads HERE
OSU’s Biochemistry/Biophysics program HERE
OSU’s College of Science HERE
Oregon State University HERE
Email Kevin Ahern / Indira Rajagopal / Taralyn Tan
The Codon Song
To the tune of “When I’m Sixty Four”
Metabolic Melodies Website HERE
Building of proteins, you oughta know
Needs amino A’s
Peptide bond catalysis in ribosomes
Triplet bases, three letter codes
Mixing and matching nucleotides
Who is keeping score?
Here is the low down
If you count codons
You'll get sixty four
Got - to - line - up - right
16-S R-N-A and
Shine Dalgarno site
You can make peptides, every size
With the proper code
Start codons positioned
In the P site place
UGA stops and AUGs go
Who could ask for more?
You know the low down
Count up the codons
There are sixty four
Recording by Tim Karplus
Lyrics by Kevin Ahern
Recording by Tim Karplus Lyrics by Kevin Ahern
7.00x Introduction to Biology or similar (undergraduate biochemistry, molecular biology, and genetics), 7.28.1x and 7.28.2x Molecular Biology or similar (advanced understanding of the central dogma)
Interested in this course for your Business or Team?
Train your employees in the most in-demand topics, with edX for Business.
About this course
In Part 3 of 7.28x, you’ll explore translation of mRNA to protein, a key part of the central dogma of biology. Do you know how RNA turnover or RNA splicing affects the outcome of translation? Although not official steps in the central dogma, the mechanisms of RNA processing strongly influence gene expression.
Are you ready to go beyond the “what" of scientific information presented in textbooks and explore how scientists deduce the details of these molecular models?
Take a behind-the-scenes look at modern molecular biology, from the classic experimental events that identified the proteins and elements involved in translation and RNA splicing to cutting-edge assays that apply the power of genome sequencing. Do you feel confident in your ability to design molecular biology experiments and interpret data from them? We've designed the assessments in this course to build your experimental design and data analysis skills.
Let’s explore the limits of our current knowledge about the translation machinery and mechanisms of RNA turnover and splicing. If you are up for the challenge, join us in 7.28.3x Molecular Biology: RNA Processing and Translation.
What you'll learn
- How to compare and contrast translation in bacteria and eukaryotes
- How to describe several mechanisms of RNA turnover and RNA splicing
- How to analyze protein structures to infer functional information
- How to design the best experiment to test a hypothesis
- How to interpret data from translation and RNA processing experiments
Week 1: Translation I – Overview and Key Players
Week 2: Translation II – Elongation
Week 3: Translation III – Initiation and Termination
Week 4: Translation IV – Regulation of Translation
Week 5: RNA Splicing I – Mechanisms
Week 6: RNA Splicing II – Proofreading and Alternative Splicing
Week 7: RNA Turnover I – Assays and General Mechanisms
Week 8: RNA Turnover II – Specific Bacterial and Eukaryotic Mechanisms
RNA can act as a carrier of information from the nucleus to the cytoplasm in the processing of protein-coding genes, as a regulatory molecule that can control gene expression, and even as an extracellular signal to coordinate trans-generational inheritance [1,2,3]. RNA binding proteins (RBPs) interact with RNA through a wide variety of primary sequence motifs and RNA structural elements to control all processing steps . Furthermore, with the increase in the number of RBPs that are becoming associated with human diseases, identifying their RNA targets and how they are regulated has become an unmet, urgent need.
To identify direct RNA targets of RBPs, RNA immunoprecipitation (RIP) and crosslinking and immunoprecipitation (CLIP) methods are frequently used. CLIP-based methods utilize UV crosslinking to covalently link an RBP with its bound RNA in live cells, enabling both stringent immunoprecipitation washes and denaturing SDS-PAGE protein gel electrophoresis and nitrocellulose membrane transfer which serves to remove background unbound RNA . Analyses of single RBP binding profiles by CLIP have provided unique insights into basic mechanisms of RNA processing, as well as identified downstream effectors that drive human diseases [5,6,7]. Further efforts to profile multiple human RBPs in the same family or regulatory function by CLIP illustrated coordinated and complex auto- and cross-regulatory interactions among RBPs and their targets [8,9,10]. Rising interest in organizing public deeply sequenced CLIP datasets to enable the community to extract novel RNA biology is apparent from newly available computational databases and integrative methods [11, 12]. However, methodological differences between CLIP approaches, combined with simple experimental variability between labs and variation in acceptable quality control metrics, add significant challenges to interpretation of differences observed.
The field of transcription regulation observed similar challenges and opportunities in integrating transcription factor target profiles . To address this challenge, the ENCODE consortium piloted large-scale profiling of transcription factor targets using a single standardized chromatin immunoprecipitation (ChIP-seq) protocol . The initial effort to profile 119 factors generated a unified dataset for creating and assaying robust quality assessment standards , and led to insights into modeling transcription factor complexes, binding modalities, and regulatory networks . More critically, however, this has served as an invaluable resource for researchers to annotate potential functional variants  and generate hypotheses across a variety of fields of interest. This success suggested that a similar effort to profile RBP targets using a standardized methodology could similarly drive significant insights in RNA biology.
To this end, we introduced the enhanced CLIP (eCLIP) methodology featuring a size-matched input control  and characterized hundreds of immunoprecipitation-grade antibodies with a standardized workflow  to generate 223 eCLIP datasets profiling targets for 150 RBPs in K562 and HepG2 cell lines . Along with orthogonal data types, this study provided insights into localized RNA processing, studied the interplay between in vitro binding motifs and RBP association (and factor-responsive targets) in live cells, and identified novel effectors of RNA stability and alternative splicing .
In this companion work, we provide further insight into how integrative analysis of RBP target profiles by eCLIP can reveal both general principles of RNA processing as well as specific mechanistic insights for individual RBPs. Although most CLIP analysis typically focuses on binding to mRNAs (both intronic and exonic), we find that for 70% of RBPs, the dominant enrichment signature is instead a variety of multicopy and non-coding elements (including structural RNAs such as ribosomal RNAs and spliceosomal snRNAs, retrotransposable and other repeat elements, and mitochondrial RNAs). These analyses can be then used to generate hypotheses about RBP function, as enrichment for the ribosomal RNA precursor corresponds with RBPs regulating ribosomal RNA maturation whereas enrichment for retrotransposable elements corresponds to both regulation of retrotransposition itself as well as suppression of improper RNA processing due to cryptic elements contained within these elements. Binding maps across meta-profiles of mRNAs and exon-intron junctions similarly show that RBP binding patterns correlate with RBP functional roles, and analysis of spliceosomal components indicates that eCLIP can be used to identify branch points and provides evidence for a 3′ splice site scanning model. In summary, these results provide further validation of the power of integrated analyses of RBP target maps generated by eCLIP in identifying novel principles of RNA biology, as well as generating RBP-specific hypothesis for further functional validation.