Who discovered RNAi and what experiment revealed it?
Craig Mello and and Andrew Fire in 1998 worked with nematodes and they discovered that dsRNA had even more gene silencing properties than ssRNA.
They also found that the dsRNA was correspondent to mature mRNA therefore it is post-transcriptionally acting.
Differentiate between the formation of miRNA and siRNA
siRNA is formed from previously existing long dsRNA molecules (these long dsRNA molecules can also be artificially administered, exogenous siRNA).
miRNAS come from RNAs transcribed in nucleus (RNA polymerase 2) and are folded and processed (DROSHA cleaves it and makes it into stem loop structure) before being exported into cytoplasm (via exportin 5) as double strand precursors.
Both go on to interact with dicer.
Explain the fate of siRNA and miRNA after interacting with DICER and how they effectively silence genes.
Both rnas interact with DICER (containing
RNAse 3 enzyme facilitating the breakdown) complex in cytoplasm to be broken down into smaller fragments (both approx. 22 nucleotides long) and 2 strands. Note that once this happens the miRNA no longer has the hairpin structure.
One strand, known as the guide strand will form the RISC (RNA induced silencing complex) complex which will guide the dsRNA to the mRNA target where complementary (full or partial) base pairing between dsRNA and mRNA occurs. Other strand (passenger strand) of dsRNA is destroyed.
siRNA has perfect base pairing with target mRNA. Once base paired, enzymes in RISC degrade the mRNA (endonucleolytic cleavage) and the gene is silenced. The cleavage occurs between 10th and 11th nucleotide of 5’ region.
miRNA has imperfect base pairing, occuring at 3’ end and seed region of mRNA (2-7 nucleotides). The imperfect base pairing allows for many potential targets whereas siRNA only has one. This also leads to degradation.
Explain how siRNA can be used to silence genes at the chromatin level.
siRNA duplexes can be loaded onto nuclear form of RISC known as RITS, which can bind homologously to chromatin to convert it into heterochromatin (condensed version that cannot actively be transcribed as transcription factors cant reach it).
What are some ways, aside from RITS, that genes can be silenced at the chromatin level?
Histone modification via acetylation, methylation, and phosphorylation.
N terminus tails of histones are targets for this, where methyl transferase can add 1-3 methyl groups to lysine or arginine.
Methylation is a characteristic of inactive heterochromatin (therefore silenced).
RITS aids this as it recruits methylating enzymes.
Briefly describe the discovery of split genes.
In an experiment where DNA was hybridized with its corresponding mRNA, electron micrographs revealed DNA is much longer , and this presented as DNA loopind where two were base paired (regions that had not pairing equivalent mRNA).
This brought forth the idea of mRNA processing, leading to discovery of introns and exons and other splicing.
Explain why introns are mainly seen only in eukaryotes.
In prokaryotes, shine-dalgarno sequence is ribosome binding site to initiate transcription, approximately 7 bp upstream.
Transcription and translation occur simultaneously, and this fast nature does not allow time for post-transcriptional modification before translation.
In addition, operons are seen in prokaryotes: continues array of reading frames yielding many mRNA transcripts coding for different proteins.
Explain the process of the addition of 5’ cap seen in eukaryotic mRNA modifications.
- Phosphate is removed from 5’ end of pre-mRNA
- GMP is added
- Methyl groups are added to guanine base at 2’ sugar position.
This cap provides protection of mRNA, and aids in transport out of the nucleus.
Ribosomes recognize this 5’ end to initiate translation.
What 3 sites does splicing of mRNA require?
5’ splice site, 3’ splice site, and the branch point (consensus sequences). Branch point lies approx 18-40 nucleotides upstream of 3’ splice site.
Explain process in which introns are removed.
They are removed in the form of a lariat in a 2 step process: 2 transesterification reactions.
1. 5’splice site cut, loops up to interact with branch point forming the lariat. The guanine of 5’ splice site interacts with adenine at branch point (phosphodiester bond broken and new one is made).
2. 3’ splice site cut and simultaneously the two exons join, lariat is released.
Lariat will eventually be cleaved at branch point to become linear and then degradation will occur.
The spliceosome facilitates this.
Explain the structure of the spliceosome and how it it assembles to facilitate splicing.
This ribonucleoprotein complex is one of largest and most complex molecular structures with approx 300 proteins and 5 small nuclear RNAs (snRNAs) each 100-200 nucleotides long: U1, U2, U4, U5, U6 which can associate with proteins and are then calls snRNPs.
- U1 attaches to 5’ splice site
- U2 attaches to branch point
- U4 U5 AND U6 join spliceosome in a complex.
- This causes 5’ end to loop over forming the lariat and U1 and U4 dissociate
- Base pairing can then occur between U6, U2 and U6 and the mRNA so all sites are now in close proximity held together by the spliceosome.
- U6 catalyses both reactions: where two exons join and lariat is release as bond breaks.
What are the 4 types of introns and where are they found?
- Self splicing (no spliceosome) located in eubacteria, eukaryotes, and bacteriophages
- Self Splicing located in eubacteria, archea, and eukaryotic organelles
- Group 3 (nuclear pre-mRNA): protein encoding genes of eukaryotes
- Group 4 (tRNA): enzymatic introns in tRNA genes of eubacteria, archaea, and eukaryotes
Explain structure of group 1 self splicing introns and the mechanism behind it.
Hairpin secondary structure with 9 stem loops necessary for splicing to occur via phosphodiester cleavage.
3’ OH of G acts as nucleophile to attack 5’ phosphate at 5’ splice site
Explain structure of group 2 self splicing introns and the mechanism behind it.
Similar to spliceosomal-introns, suggesting evolutionary relatedness.
The second structure is also a lariat.
2’ OH of adenosine in intron acts as nucleophile, attacking 5’ splice site, forming lariat.
What are some of the ways that mRNA transcription is coupled with mRNA processing?
Coupling events are mediated by CTD on large subunit of RNA polymerase 2, as the CTD recruits processing enzymes.
- The 5’ cap is added as soon as 5’ end of mRNA is transcribed and emerged from RNA pol. Capping enzymes recruited at this time to allow immediate 5’ capping.
- Assembly of the spliceosome occurs co-transcriptionally via the CTD recruiting the splicing proteins as the mRNA is still being transcribed, and assembly of spliceosome can occur on varying intron locations. Cleavage of mRNA requires Rat 1, which can degrade remaining mRNA that did not need to be transcribed.
- Polyadenylation factors recruited by CTD and RNA is cleaved at poly A site.
What are the 4 typical ways alternative splicing is achieved?
- Skipping an exon
- Intron retention
- Alternative 5’ or 3’ splice sites (varying exon lengths)
- Mutually exclusive exons
What is the result of alternative poly A cleavage sites?
Different lengths of mRNA products will be created, so different proteins sizes with differing functionality.
What might mutations about splice sites result in?
- disrupt consensus sequence preventing spliceosome binding
- Create new splice sites.
- Initiate usage of cryptic splice site.
What is familial dysautonomia (FD)?
Recessive genetic disease caused by affecting cells in autonomic nervous system (involuntary nerve system).
Result of a mutation in a gene (T instead of C) in 5’ splice site leading to the skipping of exon 20.
What is beta thalaseemia?
Mutations in b-globulin gene (component of hemoglobin) resulting in destruction of red blood cells leading to anemia. An example of mutation leading to this includes point mutation in intron 2 3’ splice site, which aborts normally splicing and leads to use of cryptic splice site.
Explain structure of tRNA.
tRNA serves as link between genetic code (mRNA) and amino acid sequence.
Amino acid attachment site is same for all tRNA molecules (CCA) and has anticodon at bottom.
Amino attachment site contacts catalytic region on large subunit allowing binding of amino acid.
Stucture of ribosome.
2 subunits: 50S and 30S to make 70S, containing 3 rRNA molecules and over 50 proteins.
3tRNA binding sites (A, P, E).
Small ribosomal subunit holds mRMA so only codon can be read by tRNA at a time.
What are the 4 steps of prokaryotic translation?
- Charging of tRNA: attachment of tRNA to amino acid, all amino acids added to 3’ adenine in acceptor stem. Once tRNA is bound to amino acid it is called aminoacyl-tRNA (super amimo acid).
Aminoacyl-tRNA synthase facilitates this, and each one is specific to the amino acid. - Initiation includes mRNA binding (Shine dalgarno sequence) to small subunit (16S rRNA). For this to happen, IF3 and IF1 hold two ribosomal subunits apart. Then initiator anticodon pairs with mRNA start codon, facilitated by IF2. This forms 30S initiation complex. If factors dissociate via GTP hydrolysis, two subunits come together to form 70S initiation complex.
- Elongation: aa-tRNA enters a site, guided by EFs and anticodon pairs. Peptidyl transferase facilitates peptide bond formation, ribosome translocation via EFs and GTP so tRNA can move into P site with new polypeptide chain, and exit via E site, opening up A site.
- Termination occurs when reaches stop codon. No aa-tRNA enters a site here, RF proteins bind to a-Site and release peptide.
What are differing characteristics in eukaryotic translation from prokaryotic translation.
- No shine dalgarno consensus sequence, instead Kozak sequence which is where start codon resides.
- 5’ Cap facilitates ribosome binding.
- Initiation complex scans mRNA until start codon is reached.
- Monocystronic (only 1 transcript and 1 protein from one translation process).
- 60S + 40 S make 80S ribosome complex.
