Lecture 11 (Linneweber) Flashcards
Alternative splicing and RNA modifications
Composition of the Genome
What do the k-value paradox, C-value paradox and N-value paradox say?
K-value paradox
- Complexity does not correlate with chromosome number
C-value paradox
- Complexity does not correlate with genome size.
N-value paradox:
- Complexity does not correlate with gene number
Mechanisms that Increase
Protein Diversity in Metazoans
Mechanisms that Increase Protein Diversity in Metazoans
- somatic DNA re-arrangement (antibody production)
- use of multiple transcription start sites
- alternative polyadenylation
- pre-mRNA editing
- alternative pre-mRNA splicing
- post-translational protein modifications
Facts on Alternative Splicing
Facts on Alternative Splicing
- Humans possess only ~26,000 protein coding genes, but there are an estimated 90,000 proteins (not counting SNPs and PTMs)
- More than 90% of human mRNAs are alternatively spliced; 85% with a minor isoform frequency of 15%
- Human-mouse comparison: alternative splicing often associated with recent exon creation/loss –> thus alternative splicing creates species-specific messages / proteins
Types of Alternative Splicing
Types of Alternative Splicing
a: Alternative 5ʹ splice-site selection
- Splicing occurs at different positions on the 5′ end of the intron, altering the start boundary of the downstream exon.
b: Alternative 3ʹ splice-site selection
- Splicing occurs at different positions on the 3′ end of the intron, altering the end boundary of the upstream exon.
c: Cassette-exon inclusion or skipping
- The central exon can either be included in the mature mRNA or skipped entirely.
d: Intron retention
- The intron can either be spliced out (left) or retained in the mature mRNA (right). If retained, the intron is treated as an exon.
Unconditional vs conditional Alternative Splicing
- Alternative splicing may be unconditional, i.e.,
two or more mRNA variants are produced in
all tissues expressing the gene. - Alternative splicing may be conditional, i.e.,
tissue specific, developmental-stage specific
or physiological-state specific.
Alternative Splicing: General
Mechanism
Cassette Exon Splicing
Cassette exon splicing
- involves the decision to include or skip a regulated exon (purple box) in the mature mRNA.
Key factors
- U1 snRNP binds the 5′ splice site, and U2 snRNP binds the 3′ splice site and branch point.
- Negative regulatory proteins (red) block splice-site recognition, leading to exon skipping.
- Positive regulatory proteins (blue) enhance spliceosome assembly, promoting exon inclusion.
Outcomes
- mRNA 1: The cassette exon is skipped, and surrounding exons are joined.
- mRNA 2: The cassette exon is included in the mature mRNA.
Muscle-Type Specific Alternative Splicing in Microgravity
Muscle-Type Specific Alternative Splicing in Microgravity
Concept
- Muscle-specific alternative splicing refers to the unique regulation of splicing in genes related to muscle proteins.
- Under microgravity conditions, such as during spaceflight, these splicing patterns are altered, which impacts muscle function and adaptation.
Key Example Titin
- Titin is a giant protein essential for the elasticity and structure of sarcomeres in muscles.
- Microgravity leads to changes in the splicing of Titin isoforms, which may affect its biomechanical properties and contribute to muscle atrophy in space.
Relevance:
- Differential alternative splicing in microgravity affects structural and functional properties of muscle proteins, leading to adaptation or dysfunction in space.
Example of Alternative Splicing
Tissue-Specific Alternative Splicing
- In the thyroid, exons 1–4 are spliced to produce calcitonin.
- In the brain, exons 1–3 and 5–6 are spliced to produce CGRP.
The kinetic model of alternative splicing
The kinetic model of alternative splicing
- Alternative splicing is influenced by the transcription elongation rate, chromatin structure, and histone modifications.
- Exons are packaged in nucleosomes, with constitutive exons (green) consistently marked by H3K36me3, ensuring their inclusion in the mRNA.
- Alternative exons (pink) may lack H3K36me3, allowing faster RNA polymerase movement and exon skipping.
- When nucleosomes packaging alternative exons contain H3K36me3, RNA polymerase slows, enabling the spliceosome to recognize and include the exon in the mature mRNA.
- Without H3K36me3, RNA polymerase transcribes rapidly, skipping the alternative exon, which is excluded from the mature mRNA.
What is RNA Editing?
RNA Editing
- Alters the RNA sequence encoded by DNA in a single-nucleotide, site-specific manner
- discovered in 1986 in Trypanosomes by Rob Benne
- >170 types of RNA modification known
What does RNA Editing lead to?
- Codon Changes
- Creation / Deletion of Splice Sites
- Extended codon recognition by tRNAs
A-to-I RNA Editing
A-to-I RNA Editing
- ADAR (Adenosine Deaminase Acting on RNA) binds to dsRNA regions.
- The adenosine (A) in the RNA is deaminated, where an amino group (-NH₂) is removed.
- This converts adenosine (A) to inosine (I).
- Inosine pairs with cytosine (C) and is interpreted as guanine (G) by the cellular machinery during translation and splicing.
- 100 Mio A-I sites in Alu elements (short interspersed nuclear elements (SINEs))
- about 200 in coding regions
Two related machineries
- ADARs: Adenosine deaminase acting on RNA
- ADATs: Adenosine deaminase acting on tRNA
Alu elements
Alu elements
- short interspersed nuclear element = SINE
- Length = ~300 bp
- Repetitive: > 1,500,000 times in the human genome
- Constitute >11% of the human genome
- Found mostly in intergenic regions and introns
- Propagate in the genome through retroposition (RNA intermediates).
- can form complementary base-pairing when they are in opposite orientations (sense and antisense)
A-to-I RNA Editing
Example GluR2 Editing
GluR2 Editing and ALS
- ADAR enzymes edit the mRNA of the GluR2 subunit of the AMPA receptor, converting adenosine (A) to inosine (I).
- This changes a codon from Glutamine (Q) to Arginine (R).
- The R form of GluR2 makes the AMPA receptor impermeable to Calcium (Ca²⁺), protecting neurons from calcium toxicity.
- Without editing, the Q form of GluR2 is expressed, making the receptor permeable to Ca²⁺.
- Excessive calcium influx damages motor neurons, leading to cell death.
- Impaired RNA editing of GluR2 has been observed in Amyotrophic Lateral Sclerosis (ALS), contributing to motor neuron degeneration. .
RNA Editing in Octopus Brains
RNA Editing in Octopus Brains
- 60% or RNAs edited vs 1% in humans
- Cephalopods have traded DNA-level flexibility (mutations and genetic evolution) for RNA-level flexibility (post-transcriptional editing). –> limits evolutionary changes at the DNA level around editing sites
Octopus RNA editing creates
- Protein diversity (multiple protein isoforms from one transcript)
- Acclimation (RNA editing in response to changes in conditions) e.g. temperature dependent editing of mRNAs for neuron excitability in poikilotherm octopus
C-to-U Editing
C-to-U Editing
- Apobec-1 enzyme binds to single-stranded RNA at a specific target sequence.
- The enzyme recognizes a “mooring sequence” near the cytidine to be edited.
- Apobec-1 deaminates cytidine (C) by removing an amino group (-NH₂).
- Cytidine (C) is converted to uracil (U).
Editing of apoB mRNA
Editing of apoB mRNA
- The Apobec-1 enzyme edits a cytidine (C) to uracil (U) at position 6666 in the ApoB mRNA.
- The editing introduces a stop codon.
- Unedited mRNA (liver): Produces full-length ApoB100, which functions in LDL transport.
- Edited mRNA (small intestine): Produces shorter ApoB48, essential for chylomicron formation and dietary fat absorption.
Mechanism
- Apobec-1 recognizes the mooring sequence near the editing site for precise targeting.
- The editing event is tissue-specific and regulated to produce distinct protein isoforms.
m6A RNA Modification
m6A RNA Modification
- m6A (N6-methyladenosine) is a chemical modification where a methyl group (-CH₃) is added to the adenine base at the N6 position in RNA.
- Writers add m6A, erasers remove it, and readers interpret it to regulate RNA stability, splicing, translation, and degradation.
- enables fine-tuned control of gene expression.
- accounts for ~50% of all methylated ribonucleotides
- More than 7,000 mRNAs in mammalian cells are
m6A-modified
- m6A exists in 0.1-0.4% of adenosines
- Occurs in: mRNA, rRNA, tRNA, small nucleolar RNAs, microRNAs, long non-coding RNAs.
- change of chemistry, no change in translation
m6A RNA Modification
Functions
Functions of m6A RNA Modification
Regulates RNA metabolism f.e. turnover
- influencing stability and degradation
- can recruit specific RNA-binding proteins (readers), such as YTHDF2, which direct the RNA to degradation pathway
Regulate stem cell maintenance and neuronal differentiation
- It marks mRNAs for selective decay or translation, controlling protein production during development.
- Ensures balance between self-renewal and differentiation in neuronal stem cells.
- Disruption of m6A leads to improper nervous system development.
miCLIP
miCLIP: Identifying m6A Sites
- m6A-modified RNA is recognized and bound by specific antibodies.
- UV light creates covalent bonds between the antibody and m6A.
- Antibody-bound RNA is isolated.
- Proteinase K Treatment Removes the antibody, leaving m6A-modified RNA intact.
- Reverse Transcription creates complementary DNA (cDNA).
- Sequencing reveals m6A locations, detected by truncations or C-to-T transitions
This method provides nucleotide-resolution mapping of m6A sites.
