what can occur at the same time and what must occur at different times during eukaryote protein formation?
before transcription is completed, RNA processing can occur (ie. 5’ capping, splicing, 3’ polyadenylation)
note: translation cannot occur before or during any of these processes because it’s in the cytosol whereas RNA processing and transcription is in the nucleus
how can we couple RNA processing and transcription?
- what is the CTD
- how does coupling occur?
hard lowkey
what is the CTD
- the C-terminal domain (CTD) is a sequence of 7 amino acids repeated 52 times in humans and is attached on to RNA polymerase II, like a tail
- it moves along the DNA with RNA polymerase II to make the mRNA strand
how is it coupled?
- During transcription elongation, the C-terminal
domain (CTD) of RNA polymerase binds RNA
processing proteins and transfers them to RNA
at the appropriate time
- the binding of RNA processing proteins occurs due to regulation of phosphorylation patterns on the CTD
- ex. two phosphorylates bind abiding by some pattern –> capping proteins bind
or
multiple phosphorylates bind abiding by some pattern –> splicing proteins proteins bind and splice out the introns on the mRNA
- The CTD is essentially the place where the proteins get ready so as soon as they need to be used by the mRNA they are available
RNA Transport Out of the Nucleus:
what are markers of mature mRNA (3) and immature mRNA (1)
- where does mature mRNA travel
- what fraction of RNA leaves the nucleus
- what degrades improperly processed mRNAs and where
- The cell selectively transports mature mRNA from the nucleus
- Markers of mature mRNA must be acquired for export
➢ cap binding complex (CBC) - some proteins on the CAP
➢ exon junction complexes (EJC) - where the introns used to be there are EJCs
➢ poly-A-binding proteins - These proteins travel with the mRNA to the cytosol
- Markers of immature mRNA must be lost for export
➢ proteins involved in RNA splicing (e.g., snRNPs)
only about 1/20 of RNA leaves the nucleus
Improperly processed mRNAs will eventually be degraded in the nucleus by the exosome.
review of translation (4)
- tRNAs match amino acids to codons (3 nucleotides) in the mRNA genetic code
- mRNA message is decoded in ribosomes made up of >50 different proteins and several RNA molecules
- Amino acids are added to the C-terminal end of the growing polypeptide chain
-Therefore, proteins are
synthesized from N- to C-terminus
why is there post transcriptional gene regulation/ mRNA quality control?
- some mRNAs are incompletely processed or damaged in the cytosol
- need to prevent production of aberrant protein which can be toxic to cells
how is mRNA quality controlled in eukaryotes?
with loops and eIFs
- Translation initiation machinery recognizes the 5’-cap and poly-A tail: (note this all occurs in the cytosol)
- Eukaryotic initiation factors (eIFs)
➢ 5’ cap bound by eIF4E
➢ poly-A binding protein bound by eIF4G - the eIF4E and eIF4E factors bind together and form an mRNA loop
- the loop, ensures that both ends of mRNA are intact (ensures it is mature mRNA so translation occurs – if no loop no translation because that means there is no cap and poly A tail i think)
- The mRNA loop recruits small a ribosomal complex (with eukaryotic initiation factors (eIFs)- next lesson) which will initiate translation at first AUG downstream of 5’ cap (some exceptions)
- The exon junction complex (EJC) also stimulates translation ensuring proper
splicing…(last marker for mature RNA and translation)
review last class: normal and abnormal splicing
- normal splicing = EJCs attached and introns spliced out
- abnormal splicing = some introns not spliced out thus EJCs not added on those introns
- this RNA needs to be degraded asap by the cytosol
mRNA Quality Control in Eukaryotes: Nonsense-Mediated mRNA Decay
Normal splicing
- after introns are spliced normally, the EJCs are added where the introns were
- The ribosome binds to the mRNA on the AUG start codon as it emerges from the nuclear pore to the cytosol
- EJCs are displaced by the moving ribosome (ribosome is the train and as it moves along the RNA from AUG to stop codon – it bulldozes the EJCs off)
- No EJCs remain bound when the ribosome reaches the stop codon exon
- mRNA is released in the cytosol
mRNA survives and there is efficient translation
mRNA Quality Control in Eukaryotes: Nonsense-Mediated mRNA Decay
Abnormal splicing
- in abnormal slicing, some introns are not spliced out and some are. for the introns that are spliced the EJC attaches as usual. For the introns that are incorrectly not spliced, EJCs do not splice
- The ribosome binds to the mRNA start codon as it emerges from the nuclear pore to the cytosol
- EJCs are displaced by the
moving ribosome (ribosome is the train and as it moves along it bulldozes the EJCs off) - There is a premature stop codon attached to every intron. as some introns are not spliced out, the stop codon is premature. thus, as the ribosome advances to the intron, it stops.
- If there are EJCs after the premature stop codon on the intron where the ribosome stops, they remain on the RNA and will not/cannot be bulldozed off – BAD!
- because of this, the EJCs calls Upf proteins to signal that there is an error and that the mRNA needs to be eliminated
- Thus, mRNA is degraded (mediated by Upf proteins)
Upf Triggers mRNA degradation
how are nonsense mediated mRNA decay (ie. the elimination of abnormally spliced mRNA) important for the evolution of eukaryotes, for immune cells, and human disease
- May have played an important role in the evolution of eukaryotes by allowing the selection of DNA rearrangements or alternative splicing patterns that produce full-length proteins
- Important role in cells of the immune systems where extensive DNA rearrangements occur to produce antibodies
- Also plays a role in many human disease cause by mutations that produce aberrant proteins. Cells can degrade aberrant mRNA and
allow functional protein to accumulate
how are mRNA quality control done in prokaryotes
Prokaryotes also have quality control for incomplete or broken mRNAs
- Ribosomes stall on broken or incomplete mRNAs and do not release – stalls because it doesn’t know what to do when the mRNA is broken
- A special RNA tmRNA (mixture of tRNA and mRNA) is recruited to the A site
- the tmRNA carries an alanine amino acid
- tmRNA acts as a tRNA by adding the Alanine attached to it onto the polypeptide (no anticodon-codon binding) (moves to the middle P site)
- After the transfer of alanine, that triggers the broken mRNA to be released and degraded by the prokaryote
- The ribosome translates 10 more codons from the tmRNA (usually 10 more alanines), which now acts as an mRNA (elongation)
- The 11 amino acid tag is recognized by proteases that degrade the entire protein (the 11 amino acid tag attached to the incomplete protein that couldnt complete due to the broken mRNA codon is degraded)
how is the degradation of abnormal mRNA different in prokaryotes and eukaryotes (2)
- In prokaryotes, exonucleases rapidly degrade most mRNAs
- In eukaryotes, mRNAs are more stable and degradation is regulated…
There are two main mechanisms: both involve gradual poly-A tail shortening
- poly A tail shortening is when an 5’-3’ exonuclease known as deadenylase (pac man protein) chops off the A’s on the 3’ tail when the mRNA reaches the cytoplasm. the tail goes from around 200 A’s to 25 A’s. This shortening acts like a timer of mRNA lifetime
- Once the poly-A tail reaches a critical length (humans = 25 nucleotides) two degradation mechanisms can occur
1st mechanism:
- continue the 3’ to 5’ degradation of the protein (chop off the rest of the 25 A’s and the protein with deadenylase and other exonucleases)
2nd mechanism
- decapping followed by rapid 5’ to 3’ degradation (instead of going through chopping the rest of the 25 A’s, just cut off the cap and rapidly degrade)
Note:
* both mechanisms can occur on the same mRNA
* cytoplasmic poly-A elongation can also occur to stabilize mRNA – ie reattachment of poly A tail
* proteins can also interfere poly-A shortening (e.g., aconitase)
explain how proteins can also interfere poly-A shortening with the example of aconitase and iron levels
- what is the receptor
- what does the receptor do upon a stimulus
- iron starvation process
- excess iron process
Another example of protein-regulated mRNA stability…
Transferrin Receptor imports iron into the cell
➢ needed when cellular iron is low (iron starvation)
➢ is not needed when cellular iron is high (excess iron)
iron starvation:
* mRNA stabilized by cytosolic aconitase protein
* binds 3’UTR
- mRNA is stable and translated
- thus, transferrin receptor is made so that iron can be imported into the cell
excess iron:
* aconitase protein binds to an excess iron molecule and undergoes a conformational change
- cannot attach to the 3’UTR mRNA due to conformational change
* mRNA released
* exposes 3’UTR endonucleolytic cleavage
site where the aconitase usually binds to on the 3’UTR. protein recognizes this sequence and knows that it needs to be degraded - thus makes a cut at thus site
- the cut causes both ends of the mRNA to degrade fast because one side doesn’t have the poly A tail and the other doesn’t have the 5’ cap.
- mRNA is degraded
- no transferrin receptor is made
how is there competition between mRNA translation and degradation in eukaryotes
look at slide 21 for diagram
- mRNA must form a loop for translation using poly A binding proteins and eIF4G and eIF4E translation factors
- the deadenylase that shortens the poly-A tail binds to the 5’ cap (like eIFs) and chops off its own tail of A’s
- these two processes are at equilibrium – the cell can control if it wants more translation (ie. inactivate deadenylase) or more degradation (ie. inactivate translation factors)
Explain Post-Transcriptional Gene Regulation with mRNA Stability and miRNAs (2)
only in eukaryotes
- eukaryotes have noncoding RNAs called micro RNAs (miRNAs) which also regulate mRNA stability (>2000 in humans)
- The primary function of miRNAs is to silence specific genes post-transcriptionally. They achieve this by base-pairing with regions of their target mRNAs, leading to their degradation and thus protein inhibition.
- they are synthesized by RNA polymerase II and initially get a 5’ cap and poly-A tail
- in the nucleus, an enzyme comes in and “crops” the double stranded miRNA, cutting off the 5’ cap and poly A tail
- then transfers into the cytosol and a Dicer enzyme dices the miRNA, leaving a small double-stranded RNA fragment
- Argonaute and other proteins come in and associate with one of the strands of the small double stranded miRNA – this forms a complex called an
RNA-induced silencing complex (RISC) - RISC seeks mRNA with complementary nucleotide sequences to the single stranded miRNA
- Two possible outcomes are possible:
1. Extensive Match: If there is extensive or near-perfect complementarity between the miRNA and its target mRNA, Argonaute within RISC slices the mRNA. This slicing leads to rapid degradation of the mRNA, effectively silencing gene expression. This process uses ATP and the RISC complex can be resued
2. Less Extensive Match: If there is less extensive or partial complementarity, Argonaute does not slice the mRNA. Instead, this imperfect pairing results in translational repression where translation of the target mRNA is blocked from the ribosome. Over time, this also leads to eventual decay of the mRNA.
what are RNA interference (RNAi) and how does it relate to miRNA
➢ Double-stranded RNAs that end up suppressing the gene expression of other RNAs in a sequence-specific manner – just like miRNA suppresses gene via degradation
➢ The proteins used in the miRNA regulatory mechanisms also serve as a
defense mechanism against foreign RNA molecules – RNAi protects the body from harmful double stranded RNA diseases via degradation
➢ Found in eukaryotes, including fungi, plants, and worms
how is there post transcriptional gene regulation with RNAi and siRNAs (2 methods)
- Many viruses (and transposable elements) produce double-stranded
RNA as part of their life cycles - RNAi destroys double stranded RNA when it enters a eukaryote, as a defense mechanism
how:
- the dicer protein complex (like those in miRNAs) cleave the double stranded RNA (RNAi) to make the small double stranded RNAs (like in miRNA). These small RNAs are called siRNAs (small interfering RNAs)
- the siRNAs can follow one of two mechanisms to degrade or repress transcription of viruses:
- siRNAs can interact with Argonaute and RISC proteins and follow the miRNA route to destroy double-stranded RNA (via slicing, ATP – flashcard 15)
or
- siRNAs can regulate transcription by forming a complex with Argonaute and RITS (RNA-induced transcriptional silencing protein). The RITS interacts with anti-parallel mRNA that is actively being transcribed by RNA polymerase 2 that is attached to the DNA sequence. If it associated with this anti-parallel strand, then it knows that this RNA being created is coming from a DNA that makes viral RNA and thus recruits chromatin modifying
enzymes to compress the DNA strand so that transcription is repressed.
in only prokaryotes, explain CRISPR Cas immunity
- Viral DNAs complementary to CRISPR regions are directed for degradation by Cas (CRISPR-associated) proteins as a degradation mechanism
how?
* short fragments of viral DNA integrate into the CRISPR locus of the DNA genome and become templates to produce crRNAs (CRISPR RNAs)
* the DNA CRISPR locus is transcribed to pre-crRNA
* pre-crRNA fragments will associate with the Cas protein making crRNAs
* now in the future if a new viral DNA infection penetrates the bacteria and matches with one of the historical DNA sequences from previous infections that were integrated into the locus and a crRNA protein, the crRNA protein will cut the double stranded viral DNA for degradation
- this process does not use antibodies, but uses memory from previous infections to chop up the RNA inhibiting translation
- similar to Argonaute (for eukaryotes), here CRISPR (for prokaryotes
- argonaute is uses RNA to cut RNA, CAS uses RNA to cut DNA
how are scientists trying to use prokaryotic Cas/CRISPR in eukaryotes
- they are trying to integrate the same technology in eukaryotes
- take RNA sequences and CAS proteins and put in eukaryotes
- that will chop the eukaryotic viral DNA
