Lecture 6 (Murray) Flashcards
Epitranscriptomics in S. pombe
Structure of the tRNA
Structure of tRNA
- Shape: Cloverleaf (2D) and L-shaped (3D).
Key regions
- Acceptor stem: CCA end binds amino acids.
- Anticodon loop: Recognizes codons on mRNA
- Antiparallel pairing: The mRNA codon is read in the 5’ → 3’ direction, while the anticodon in tRNA pairs in the opposite, 3’ → 5’ direction
- D loop, T loop, and variable loop: Provide structural support and function.
Function of the tRNA
Function of tRNA
- Aminoacylation: Charged by aminoacyl-tRNA synthetase (aaRS).
- CCA end: Binds amino acids and forms peptide bonds during translation.
Role in translation
- Moves through ribosome’s A, P, and E sites.
- Facilitates polypeptide chain elongation.
- Modifications: Enhance stability and translation accuracy.
tRNA Modifications
tRNA Modifications
- 13 Modifications per tRNA.
- Position 37: Prevents frame shifting (slipping on mRNA).
- Position 34: Enables wobble base pairing with codon’s 3rd position (Allows one tRNA to read multiple codons & reduces tRNA diversity requirement)
- Function: Enhances fidelity, prevents slippage, maintains reading frame.
Example Modifications of the Anticodon
Example Modifications of the Anticodon
Restriction
- A “U” can pair with every base (four-way wobbling)
- Restriction of tRNA by modification of “U” will lead to pairing with only 2 bases
Expansion
- Expand decoding possibility for tRNA with “A” which can only basepair with “U” -
- Base modification lets “A” basepair with 3 bases
Dnmt2 Enzyme
Dnmt2 Enzyme
- Homologous to other DNA methyltransferases but does not methylate DNA.
- Present in S. pombe (Pmt1) but absent in S. cerevisiae (budding yeast)
- Functions as a tRNA methyltransferase, specifically methylating cytosine at position 38 in tRNA.
- Requires prior incorporation of queuine (similar to guanine) by eTGT at position 34 by to stimulate methylation by Dnmt2.
- Queuine Source: Not synthesized by eukaryotes; must be obtained from diet.
- Importance: Stabilizes tRNA and improves translation fidelity.
Biosynthesis of queuosine in bacteria
Queuosine Biosynthesis (Bacteria)
- Pathway: GTP → DHNTP → CPH₄ → CDG → preQ₀ → preQ₁ → Queuosine.
Enzymes:
- bTGT: tRNA guanine transglycosylase Incorporates preQ₁ into tRNA (position 34).
- QueA: Converts preQ₁-tRNA into queuosine-tRNA.
Biosynthesis of queuosine in eukaryotes
Biosynthesis of queuosine in eukaryotes
- Eukaryotes salvage queuine from diet or external tRNA.
- These external tRNAs are digested, releasing free queuine
- Queuine is incorporated into tRNA by eTGT (Qtr1 and Qtr2 in S. pombe) by replacing Guanine at position 34
- Enhances translation efficiency and accuracy by stabilizing the wobble position.
Effect of queuosine on eukaryotic translation
Effect of queuosine on eukaryotic translation
- Enhances wobble base pairing; increases translation efficiency but is not essential.
- Ribosome stalls without Q → protein misfolding → aggregation.
- Misfolding exposes hydrophobic regions → protein clumping and functional loss.
- Equilibrates translational speed with reduced protein misfolding.
Detection methods for queuosine modification
Boronate Northern Gel
Boronate Northern Gel
- Separates Q-tRNA from G-tRNA.
- Boronate binds to queuosine’s ribose.
- Example: tRNA-Asp in S. pombe.
Detection
- Without queuine (-): Only G-tRNA detected.
- With queuine (+q/+Q): Q-tRNA appears as a shifted band.
- Probes: Radiolabeled/fluorescent probes specific to tRNA enable accurate detection.
Detection methods for queuosine modification
LC-MS/ MS
LC-MS/ MS
- Detects queuosine in tRNAs with high sensitivity.
- tRNA digested, fragments separated by LC.
- MS1 measures mass, MS2 confirms structure.
- Queuosine detected in S. pombe tRNA-Asp.
- Sensitive, confirms structure, quantitative.
Detection methods for queuosine modification
Nanopore sequencing of tRNAs
Nanopore sequencing of tRNAs
- Preperation: Ligate splint adapter to 5’ end and RMX adapter to 3’ end; elongate tRNA for nanopore sequencing.
- RNA passes through a biological pore under applied voltage; voltage changes (current signals) are measured.
- Each nucleotide produces a unique current signal influenced by its base and neighboring sequence.
- Detects standard and non-standard bases, but signal analysis is complex due to multiple bases in the pore.
- Modifications increase error rates, often indicating non-standard bases.
- Reads are aligned to reference sequences; differences highlight tRNA modifications.
Detection methods for queuosine modification
Q-Mutational-Profiling–Seq: Q-MaP-Seq
Q-MaP-Seq
- Detects queuosine via reverse transcription with RT-KTQ M1.
- Reverse transcriptase misincorporates nucleotides at Q sites.
- PCR amplifies misincorporations; NGS identifies Q-modified sites.
- Misincorporation rates indicate queuosine presence.
Detection methods for queuosine modification
Q-RIP-Seq
Q-RIP-Seq
- Q-RIP-Seq: Detects Q-modified RNAs via metabolic labeling.
- Uses preQ₁-L5 (precursor with azide group).
- Click reaction links biotin to Q-labeled RNA.
- Purified using streptavidin-coated magnetic beads.
- Sequencing identifies Q modifications by comparing WT and mutant data.
Explain the term „Epitranscriptomics“
End of Lecture Question
Epitranscriptomics
- Refers to the study of chemical modifications on RNA molecules (e.g., mRNA, tRNA, rRNA) that regulate their function, stability, localization, or translation without altering the underlying RNA sequence. These modifications act as an additional regulatory layer, much like epigenetics does for DNA.
How do tRNA modifications affect tRNA function?
End of Lecture Question
tRNA modifications enhance tRNA function by:
- Stabilizing tRNA and Preventing Errors: Modifications maintain proper tRNA structure and prevent frame shifting, enhancing codon-anticodon interactions and decoding accuracy.
- Expanding Decoding Flexibility: Modifications like queuosine (Q) enable wobble base pairing, allowing tRNA to recognize additional codons efficiently.
- Ensuring Translational Efficiency: Modifications regulate translational speed to prevent ribosome stalling and reduce the risk of protein misfolding and aggregation.
How do tRNA modifications affect decoding?
End of Lecture Question
tRNA modifications affect decoding by
- Expanding Codon Recognition and Balancing Accuracy: Modifications at position 34 (wobble position) expand the tRNA’s ability to recognize multiple codons for the same amino acid while maintaining decoding accuracy.
- Enhancing Stability and Accuracy: Modifications strengthen anticodon-codon interactions, increasing accuracy and reducing errors like frame shifting.
- Optimizing Translational Speed: Modifications ensure smooth ribosome movement, preventing stalling during translation.
Describe how LC/ MS can be used to detect tRNA
modifications! Pros and cons?
End of Lecture Question
How LC-MS/MS Detects tRNA Modifications
- Sample Preparation: tRNA is isolated and enzymatically digested into smaller fragments, such as nucleosides.
- Liquid Chromatography (LC): Fragments are separated based on chemical properties (e.g., polarity, charge).
Mass Spectrometry (MS/MS):
- MS1: Measures the molecular mass of each fragment to identify nucleosides.
- MS2: Fragmentation provides structural details, confirming the presence of specific modifications.
- Data Analysis: Differences in mass and fragmentation patterns are used to detect and quantify tRNA modifications.
Pros
- High Sensitivity: Can detect modifications present at low abundance in tRNA.
- High Specificity: Provides detailed information about the type and location of modifications.
- Quantitative Analysis: Measures the degree of modification across different samples, enabling comparative studies.
Cons
- No Full Sequence Identification: Cannot determine the complete sequence of the tRNA, only fragments or nucleosides.
- Low Throughput: Slower compared to high-throughput methods like sequencing.
- Expensive and Specialized: Demands advanced instrumentation and technical expertise.
