1-16 Eukaryotic Transcription II Flashcards
What happens to eukaryotic mRNA immediately following transcription?
Eukaryotes covalently process mRNA and transport it out of the nucleus into the cytoplasm for translation. Some covalent processing modifications include 5’-capping, 3’-polyadenylation, splicing, and RNA editing; 5’-capping and 3’-poly (A) are added co-transcriptionally.
The eukaryotic procedure is very different from the procedure of prokaryotes, who translate all mRNA co-transcriptionally.
What covalent modifications are necessary for mRNA translation in eukaryotes, and why?
5’-capping and 3’-poly (A) tail, which are covalently processed co-transcriptionally. Proteins that bind to 5’-caps and 3’-poly (A) tails serve the following functions:
- Stabilize mRNA by protecting it from degradation
- Facilitate nuclear export
- Allow translation by ribosome
What are the consequences of introns?
- Alternative splicing: expand repertoire of gene products
- Increased homologuous recombination: expands target size of genes
- Possible regulatory advantage: splicing is regulated, like transcription
- “Selfish DNA”: remnants of transposon invaders
What is required for intron removal?
3 mRNA sequences, which generally recruit proteins that direct splicing:
- 5’ donor site
- Branchpoint
- 3’ acceptor site
What is the mechanism of pre-mRNA splicing?
- Before the mRNA leaves the nucleus, the branchpoint binding protein (BBP) recognizes and binds to the branchpoint sequence, while snRNPs do the same for the 5’ donor and 3’ acceptor sites.
- Various snRNP rearrangements bend the mRNA.
- A conserved (A) in the intron attacks the 5’ splice site, cutting the sugar-phosphate backbone and forming a lariat.
- The spliceosome rejoins the 5’ end of exon 1 with the 3’ hydroxyl group of exon 2, linking exons 1 and 2 and releasing the lariat.
At the protein level, what is the result of alternative splicing?
Proteins encoded by different spliced mRNAs may have different amino acid sequences, depending on which coding exons are included.
Most human genes produce at least 6 different alternative spliced mRNAs, a phenomenon most prominent in the nervous system.
What are some features important for splicing regulation, and why should we study them?
- Cis-regulatory sequences
- Intron/exon lengths
- Nucleotide composition
- RNA secondary structure
- Nucleosome positioning
- Chromatin modification
Understanding this “splicing code” is important to predict splicing pattern variations associated with disease, and engineering it may help develop therapeutic treatments.
What is RNA interference?
A process by which silencing RNAs (siRNAs) and micro RNAs (miRNAs) are incorporated into natural cell pathways to inhibit gene expression, either by translation inhibition or mRNA destruction.
What are the three most common small RNA types, how long are they, and what do they do?
Micro RNAs, silencing mRNAs, and piwi-interacting mRNAs all use RNA-RNA interactions to silence genes. They are typically 20-25 nt and can potentially target multiple mRNAs.
- miRNAs repress translation
- siRNAs cleave target mRNAs
- piRNAs keep transposons silent in germline
What are long non-coding RNAs (lncRNAs)?
lncRNAs are typically >200 nt and engage in RNA-RNA, RNA-DNA, and RNA-protein interactions to regulate gene expression. They often recruit proteins to modify chromatin, can act in cis or trans, and can target multiple genes.
What is a transcriptome?
A transcriptome is the set of ALL RNA molecules transcribed in one cell or a population of cells.
What is RNA-seq?
A method that measures ALL RNAs in a cell and their individual abundance of expression (based on the number of “reads”).
What are eQTLs, and why are they important?
Expression Quantitative Trait Loci (eQTLs) use RNA-seq capabilities to quantify all RNAs in a cell and find changes in individual RNA expression and mechanisms that differentiate normal from diseased cells (as opposed to variations between tissues/individuals).
- eQTLs are heritable and can be mapped
- eQTLs can occur for both coding and non-coding RNAs
- eQTLs can act in cis or trans
What are GWAS studies, and what have they demonstrated thus far?
Genome wide-association studies (GWAS) identify genomic variations that are linked to diseases/traits. >90% of disease-/trait-related variants map to non-coding DNA regions.
