RNA Types and Processes PDF

RNA Types and Processes Functional Transcription/Translation: Involves RNA types like mRNA, tRNA, and rRNA that directly participate in synthesizing proteins. Mostly Regulation Transcription/Translation: Involves RNA types like snRNA, miRNA, siRNA, and snoRNA that regulate gene expression and RNA pr...

RNA Types and Processes Functional Transcription/Translation: Involves RNA types like mRNA, tRNA, and rRNA that directly participate in synthesizing proteins. Mostly Regulation Transcription/Translation: Involves RNA types like snRNA, miRNA, siRNA, and snoRNA that regulate gene expression and RNA processing, rather than being directly involved in translating proteins. Role of snRNA in Intron Splicing Intron splicing is important because it removes non-coding parts (introns) from pre- mRNA to make the final product (mature mRNA) that can be used for protein making. How do They Know Where to Cut? Consensus Sequences:  At the ends of each intron, there are specific consensus sequences (like special signs) that signal where to cut.  These sequences help the splicing machinery know where to do its job. 5' and 3' Splice Sites:  The 5' splice site (the start of the intron) has a GU sequence, and the 3' splice site (the end of the intron) has an AG sequence.  Due to their importance, these sequences are highly-conserved and are found in almost all genes with introns. Think of them as “cut here” signs. snRNAs:  snRNAs combine with proteins to form spliceosomes  The snRNA in these spliceosomes recognizes the GU and AG sequences at the splice sites.  They guide the spliceosome to the right spots to cut the introns out and connect the exons together. More on Spliceosomes What are Spliceosomes made of?: Small nuclear ribonucleoprotein particles (snRNPs) What are snRNPs?  snRNAs that are U rich, hence the name U1, U2, U3 to U6, etc.  Proteins - 100 different proteins can be associated with these particles. Spliceosomes: Are assembled from 5 snRNPs and their pre-mRNA. How Do the snRNAs in the Spliceosomes Aid in Splicing? 1. Key Interactions: o The U1 snRNA interacts with the 5' splice site (the beginning of the intron) and helps mark where the splicing should occur. o The U2 snRNA binds to a specific region known as the branch-point sequence (this is often an adenine nucleotide referred to as branch-point A). 2. Importance of Interactions: o The interactions between U1 and U2 snRNAs are essential for recognizing where the cuts should happen in the pre-mRNA. RNA Decay/Degradation Cytoplasmic mechanisms of Post-transcriptional control Until now, regulation of gene expression was traditionally believed to occur predominantly through transcriptional control We now know that there are other ways the cell controls mRNA, the RNA that is made from DNA, after it’s created. Stable vs Fleeting:  Many prokaryotic mRNAs are highly stable and can exist in high concentrations in the cell cytoplasm. This means they can produce lots of proteins.  Other mRNAs, like those in eukaryotes, are fleeting. This results in bursts of protein expression Translational Silencing  Sometimes, the cell needs to control how much protein is made from mRNA without breaking down the mRNA itself.  This control is called translational silencing. Transient mRNA  For transient mRNAs, their life is mainly controlled by degradation. How is this controlled:  The cap (a special structure at the beginning of the mRNA) and the tail (a chain of nucleotides at the end) are very important for keeping mRNA stable.  If one or both of these are removed, the mRNA will degrade. Degradation Pathways and Machinery The Degradation Machinery  The degradation machinery (the group of proteins that do the breaking down) is drawn to mRNAs that have lost their polyA tail (the long stretch of adenine nucleotides at the end of the mRNA). When the tail is shortened, the mRNA is marked for destruction.  The mRNA can be degraded in two directions:  o 5' to 3' direction o 3' to 5' direction Pathway (a): Deadenylation-dependent mRNA decay  MOST mRNAs undergo decay by this pathway.  Shortening the PolyA Tail: Enzymes called deadenylases gradually shorten the polyA tail. This makes the mRNA more vulnerable to degradation.  CCR4 Mechanism: Most mRNAs are degraded by a mechanism that involves an exonuclease called CCR4, which helps remove the tail. Additional Steps in Pathway (a)  Rapid Degradation: Some mRNAs are marked for quick breakdown by special proteins that target them for faster destruction.  5′ to 3′ Decay: o In this direction, a complex called Lsm1–7 attaches to the end of the mRNA and causes the cap at the front to be removed (this process is called decapping). o This decapping is done by a pair of proteins known as the DCP1–DCP2 complex. After the cap is removed, a 5’-3’ exoribonuclease called XRN1 can start breaking down the mRNA from the front end.  3′ to 5′ Decay: o For this direction, a multi-protein complex called the exosome does the degrading. This complex can break down various types of RNA. o After the exosome works on the mRNA, another enzyme called DcpS removes the cap to finish the decay process. Pathway (b): Deadenylation-Independent mRNA Decay  Some mRNAs don’t follow this usual process of losing their polyA tail first.  These are called deadenylation-independent mRNAs because they can be degraded without losing their tail first. How Does It Work? o Example: a type of yeast called Saccharomyces cerevisiae 2. Blocked Tail: o In these cases, the polyA tail is somehow protected, meaning enzymes that normally shorten the tail (called deadenylases) can’t get to it. This keeps the tail intact. 3. Recruiting the Decapping Machinery: o Since the tail is protected and can’t be removed, the cell uses a different method. o It recruits the machinery that removes the cap at the front of the mRNA instead.  o An enhancer of decapping-3 called Edc3 helps in this process. It binds to a special part of the mRNA (called a decay-inducing regulatory element) that signals for the decapping machinery to come in. 4. Decapping and Degradation: o The decapping machinery (specifically the DCP1/DCP2 complex) removes the cap at the front of the mRNA. o After the cap is removed, the mRNA can then be degraded by another enzyme called XRN1 Pathway (c): Endonuclease-Mediated mRNA Decay  Cleaves mRNA, generating 2 fragments that both have 1 unprotected end.  The unprotected ends leave the fragment vulnerable to XRN1 and the exosome.  This is probably the most efficient way to degrade mRNA.  Cells have several endonucleases that can target specific mRNAs, making this a powerful way to control mRNA levels.  Some of these endonucleases are even found in the nucleolus, where they can help process other types of RNA like rRNA. RNA Interference MicroRNA/miRNA  Small ssRNA fragments, approx. 21-25 nucleotides long.  Function as post-transcriptional regulators  Scientists think miRNAs target around 50-60% of all genes in mammals, so they play a huge role in gene regulation.  Most are cellular (some circulating).  Function in RNA silencing. RNA silencing helps cells control which proteins get made and how much. How Does miRNA Work? 1. Base-Pairing: o miRNA binds to an mRNA molecule by matching up with part of its sequence (kind of like a lock and key). o In plants, this match is almost perfect, so the miRNA and mRNA fit very tightly. o In animals, it doesn’t need to be perfect—just 6-8 matching nucleotides are enough to "seed" the connection. 2. Effects of miRNA Binding:  Once the miRNA attaches to its target mRNA, it can stop the mRNA from making proteins in a few ways: o Cutting: The mRNA gets cut and broken down. o Destabilizing (Deadenylation): The polyA tail (which protects the mRNA) gets shortened, making the mRNA easier to degrade. o Repressing Translation: The mRNA is prevented from being read by the cell’s protein-making machinery, so no protein is made. Synthesis of miRNAs in animals 1. miRNA genes are transcribed , resulting in a primary miRNA (pri-miRNA). 2. Processing in the Nucleus with Drosha: a. In the nucleus, a class 2 RNase III enzyme called Drosha, processes the pri- miRNA into a pre-miRNA (hairpin) 3. Exportin-5 (EXP-5) mediate the transport of pre-miRNAs to the cytoplasm. 4. In the cytoplasm: a. Dicer, an RNase III type protein, cleaves the pre-miRNA and removes the the Hairpin. b. Now, the 70 bp pre-miRNAs are mature miRNAs (21-25 bp). 5. Loading onto Argonaute and Forming RISC: a. The mature miRNA attaches to a protein called Argonaute (AGO2). b. Argonaute selects one strand of the miRNA to keep (the guide strand) and discards the other strand.  c. Together, the guide strand and Argonaute make a complex called RISC (RNA-Induced Silencing Complex), which is now ready to go find matching mRNAs and silence them. Synthesis of miRNAs in plants  In plants, the entire process of cutting down the pri-miRNA to a mature miRNA happens in the nucleus (no Exportin-5 is needed).  Plants use an enzyme called Dicer-like 1 (DCL1), which does all the cutting steps.  Once the miRNA is fully processed, it’s sent out of the nucleus and binds to Argonaute proteins in the cytoplasm to form the RISC complex, just like in animals. Actions of miRNA Gene silencing may occur either via mRNA degradation or preventing mRNA from being translated. The Regulatory Mechanism Process is determined by the complementarity of the miRNA to the target mRNA. Complete Complementarity:  If the miRNA perfectly matches the target mRNA, AGO2 cleaves the mRNA directly.  This initiates its degradation, leading to the mRNA being broken down through deadenylation, decapping, and exonuclease activity Imperfect Complementarity:  If the miRNA only partially matches the mRNA (usually matches about 6-8 nucleotides in the 5’ “seed” region), it doesn’t destroy the mRNA.  If the miRNA-RISC complex binds to the 3’ UTR of complementary mRNAs, it blocks/represses translation.  The exact mechanisms of translation repression via miRISC is unknown. Are miRNAs Relevant to Life? Yep miRNAs Control Important Functions:  miRNAs are tiny pieces of RNA that help regulate how many proteins are made from certain genes in mammals, including humans. Because they’re so important in controlling proteins, they’re therapeutic targets. miRNAs Are Universally Important:  These miRNAs are highly conserved, meaning they’re very similar across different animals, showing they’ve been important in evolution.  They play roles in development and regulate between 60-90% of all human genes that code for proteins (functional). Diseases from miRNA Imbalance:  If miRNAs are dysregulated, it can lead to diseases. For example, some cancers and brain disorders like Alzheimer’s, Parkinson’s, and Huntington’s can be linked to miRNA issues. Therapeutic Approaches Using miRNAs:  miRNA Mimics: Scientists can create synthetic (man-made) miRNAs to mimic natural ones. These can be added to cells to help lower the activity of harmful genes and bring things back to normal.  miRNA Inhibitors: Sometimes, we want more of a certain beneficial protein. To do this, scientists use miRNA inhibitors (steric blockers), which stick to the miRNA and stop it from doing its usual job. This can lead to an increase in helpful proteins in the body. Small Interfering RNAs/siRNAs  Small, single-stranded ds oligomers, 21-22 bp.  Down-regulate gene expression based on complementarity to the target mRNA.  Can be used to block synthesis of disease-causing proteins.  Unlike miRNAs, siRNAs are highly specific.  Function in the protection of genome integrity.  Respond to foreign nucleic acids like viruses (innate immunity).  In plant cells, the siRISC (siRNA and protein complex) directs heterochromatin formation by: o Associating with new (nascent) RNA and RNA polymerases. o This can lead to activation of DNA methyltransferase(DMT) that methylates DNA. o This results in the DNA forming heterochromatin. o Heterochromatin is tightly packed, inactive DNA.  In mammals, a similar job is done by long non-coding RNAs (lncRNAs), which are like longer pieces of RNA (~200bases long). siRNA Synthesis 1. Begins as a long dsRNA. 2. Drosha cleaves this strand into hairpin structures/pre-siRNA fragments. 3. These fragments are further cleaved by Dicer (hairpin is removed). 4. Ago binds to this mature siRNA and then binds to RISC forming a complex. 5. A guide strand is selected and the other strand is discarded. It’s now a single-stranded siRNA. 6. The guide strand directs the RISC to a perfectly complementary RNA target, which is then degraded. siRNA vs miRNA siRNA miRNA Externally sourced Internally sourced Derived from longer RNA products Shorter RNA products High complementarity is required Only requires a seed of 6-8 nucleotides (specific targeting) (broad targeting) More likely to cleave More likely to repress translation  Both use Drosha, Dicer and Ago  Same mechanisms of mRNA degradation  Both siRNA and miRNA mimics are ~21 nucleotides RNA duplexes inducing mRNA silencing Improving gene-silencing efficiency with siRNA:  Longer dsRNA boosts potency but can activate the immune system, so smaller, processed siRNA is preferred.  Targeting specific mRNA regions enhances silencing, as certain spots work better.  Guide strand accuracy is crucial to avoid cutting the wrong sequences.  Using minimal siRNA concentrations prevents off-target effects.  Avoid miRNA-like effects by skipping miRNA seed sequences in siRNA design.  Multiple siRNAs targeting the same mRNA increase the likelihood of effective silencing. Piwi RNA  One of the most abundant small RNAs in the mammalian cell  Larger than miRNAs (21-35 nucleotides long)  Not as conserved as miRNA’s and much more complex  piRNA partners with proteins called piwi proteins to form a piRNA-piwi complex. Unlike miRNA, they don’t need the "Dicer" enzyme to be made (Dicer independent). Function:  Maintain genome integrity by silencing transposable elements. transposable elements, also called "jumping genes.", are bits of DNA that can randomly move around, which could cause dangerous mutations if not controlled. How?: How do they silence transposable elements?  The piRNA-piwi complex forms RISC complexes which cleaves transposon and retrotransposon RNA More on piRNA: 1. piRNA regulates chromatin structure via methylation. a. When transposon RNA is tightly packed forming heterochromatin, it becomes inactive (silenced). 2. piRNA in the Testes: a. piRNA is especially active in germ cells (sperm) in order to prevent transposons being passed onto the next generation. Bacterial Small RNAs (sRNAs):  Quantity: Bacteria have hundreds (not thousands) of small RNAs (sRNAs). Types of sRNAs: 1. Anti-sense sRNAs: These are synthesised from the complementary DNA of the gene they regulate. Their main role is to repress genes that code for potentially toxic proteins. 2. Base-pairing sRNAs (limited complementarity): These sRNAs the bacterial version of miRNAs and siRNAs. Modulate mRNA stability and translation. Generated as unprocessed single entities (approx. 100 bases), and they need a "seed region" of at least 6-8 bps to attach and regulate the mRNA.  Multi-Function sRNAs: Some sRNAs can do more than one job—they can also code for proteins or function as ribozymes. snoRNAs Location & Size: snoRNAs are small RNAs, about 60 to 300 bases long, and are found only in the nucleolus (a part of the cell’s nucleus). Main Job: snoRNAs guide site specific post-transcriptional modification of pre-rRNA pre- tRNA’s and pre-snRNAs, Why These Changes Matter: These modifications mostly happen in parts of the RNA that are "highly conserved," meaning they are important and have stayed the same across many species. This suggests these changes are essential for these RNAs to do their jobs, especially for protein-making in ribosomes. Two Main Types of snoRNAs: 1. Box C/D snoRNAs: These are involved in adding methyl groups to the RNA, which is like adding tiny "tags" that help stabilize the RNA. 2. Box H/ACA snoRNAs: These help change certain RNA bases into a slightly different form called pseudouridine, which also helps the RNA keep its shape and function properly.  "Orphan" snoRNAs: There are some snoRNAs, called "orphans," that don’t have an obvious function yet—they don’t seem to have any specific target or purpose we've identified. What Do snoRNAs Actually Do? 1. Two Key Modifications: snoRNAs guide two main changes to other RNAs: o Methylation: Box C/D snoRNAs add a small chemical group (a methyl group) onto the 2’O ribose part of the RNA. o Pseudouridylation: Box H/ACA snoRNAs change certain parts of the RNA into a form called pseudouridine, helping the RNA stay stable and function well. 2. How They Find Their Spot: Each snoRNA matches up briefly (like a temporary "lock and key") with specific parts of pre-rRNA or pre-tRNA to make sure the modification happens in the right place. 3. Partnering with snoRNPs: For snoRNAs to work, they need to bind to proteins called snoRNPs (small nucleolar ribonucleoproteins). Together, they form a complex that can make these precise modifications to RNA. Other snoRNA Functions  Helping in Alternative Splicing: Some snoRNAs help to produce shorter versions of RNA by cutting out certain parts. This "alternative splicing" creates different forms of proteins from the same gene.   Creating Small Regulatory RNAs: Some snoRNAs evolve into small RNAs that look and act similar across species. Some of these can even attach to AGO2, a protein involved in gene silencing, hinting that they might help control other genes.  Link to Diseases: When snoRNAs are (dysregulated), it’s linked to certain diseases. Scientists are interested in these snoRNAs as possible biomarkers, meaning they could help diagnose diseases or show disease progression. scaRNAs  scaRNAs: Small Cajal body-specific RNAs, a subtype of snoRNAs, found in the Cajal body within the cell nucleus.  Function: Aid in the production of snRNPs ("snurps") which are crucial for RNA splicing.  Modification: Guide methylation and pseudouridylation of specific snRNAs (U1, U2, U4, U5, and U12).  Purpose: Ensure snRNPs are properly modified to improve RNA splicing efficiency. Riboswitches  Definition: Riboswitches are RNA sequences found in bacteria that help regulate gene expression without needing proteins. They serve as an alternative to traditional transcriptional regulation mechanisms, like operons. Location:  Found in the 5' untranslated region (5' UTR) of bacterial mRNAs. This area is located just before the start codon of the mRNA.  They are part of the mRNA itself, meaning they are cis-acting elements—they influence the same RNA molecule they are part of. Function:  Riboswitches bind small molecules, such as metabolites or metal ions, acting as ligands. When these molecules bind, they trigger changes in the structure of the mRNA. Aptamers What They Are: Aptamers are special RNA molecules that can fold into unique shapes, almost like how proteins and antibodies do. These shapes allow them to bind to other molecules, such as proteins or small chemicals, very selectively. How They Work: When an aptamer binds to a molecule (called a ligand), it can change its shape. This shape change can affect gene expression, usually by lowering the amount of protein made from a gene. Types of Riboswitches:  Translational Riboswitches: These control gene expression by changing how accessible the ribosome binding site or start codon is, which affects how proteins are made.  Transcription-Regulating Riboswitches: These can create structures that stop the transcription process (the copying of DNA to RNA) by making the RNA unstable. This stops the RNA polymerase (the enzyme that makes RNA) from working effectively. Other Functions: Some aptamers can cause the mRNA (the messenger RNA that carries instructions for making proteins) to be broken down, change how its ends are processed, or even act as their own enzymes in response to binding with a ligand. Mechanism:  When the concentration of the relevant metabolite (in this case, guanine) is low (- M), the riboswitch structure allows the RNA to continue transcription, meaning the genes can be expressed.  Conversely, when the metabolite concentration is high (M+), it causes the riboswitch to change its shape, which leads to transcription termination. This means the expression of those genes is turned off when there is enough guanine present, preventing unnecessary production of enzymes. Example:  A classic example of a riboswitch is found in the Bacillus subtilis xpt-pbuX operon. This operon encodes enzymes necessary for purine synthesis.  In Bacillus subtilis, this riboswitch motif appears in at least five different transcriptional units, which together encode 17 genes involved in purine transport and biosynthesis.