BIO230 lecture 6 notes
- Quality control of mRNA in the cytosol IN EUKARYOTES:
Only about 1/20 mRNA molecules make it out of the nucleus. Some mRNAs are incompletely
processed or damaged when they come out to the cytosol. The cell needs to prevent these
mRNAs from being translated because they can produce aberrant proteins that have toxic
effects on cells (they can be sticky and either induce or interfere cellular processes that can
hurt the cell)
First let’s look at translation because a lot of these quality control mechanisms are tightly
coupled with translation:
tRNA molecules match amino acids to specific codons in the mRNA; codons are 3
nucleotides. The process of translating those 3 nucleotides into specific amino acids is
called the genetic code. 3 codons do not encode for amino acids: they indicate a stop of
translation. There is also one codon for methionine which serves as a start codon for
This translation occurs on large complexes called ribosomes. Ribosomes are a mixture of
proteins and RNA; almost 50 different proteins and RNA molecules make it up. The
ribosome is divided into a large and small subunit that come together on the mRNA for
protein synthesis. There are 3 sites in the ribosome, A site (amino acid site) where the
tRNA with the free amino acid goes, the P site (peptide site) where the tRNA with the
peptide associated with it stays, and the E site (exit site) where the tRNA will leave the
A specific tRNA with a specific amino acid attached to it comes in, matches to a
codon at the A site, and transfers the amino acid onto the growing peptide chain to
produce a peptide bond (note that the amino acid is added to the C-terminus of the
growing polypeptide chain that is why protein synthesis occurs from the N-
terminus to the C-terminus from the 5’ end to the 3’ end of the mRNA).
Recall that initiation of translation requires eukaryotic initiation factors that need to
come together for the ribosome to start translating. These initiation factors bind to the
5’ cap and the poly-A binding proteins that are bound to the poly-A tail of the mRNA.
There are 2 specific eukaryotic initiation factors that bind: EIF4E (displaces the cap
binding complex that is bound to the 5’ cap) and EIF4G (it binds to the poly-A binding
proteins that bind to the poly-A tail. This binding is a sign that the mRNA has been
processed properly and that the mRNA molecule is intact; damaged mRNA won’t have
both the 5’ cap and the poly-A tail. After this, there is the recruitment of the small
ribosomal complex, which eventually initiates translation at the first start codon, AUG.
There are some exceptions where this first codon will be skipped and the second
one will be used this is called leaky scanning
The exon junction complex (EJC) also stimulates translation; it has a critical function in
an important quality control mechanism called nonsense-mediated mRNA decay. It is
based on the recognition of premature stop codons in the mRNA molecules, and these
premature stop codons are called nonsense mutations because it shifts the protein and
you get a stop codon before translation should actually stop. This often occurs if there is improper splicing; it shifts the frame of the mRNA molecule and this introduces a stop
codon where it doesn’t belong.
Normal situation: As the mRNA molecule leaves the nucleus, a ribosome will bind to
it as it emerges from the nuclear pore and do a “test round” of translation. As it is
doing so, it is translating the mRNA and it removes/displaces the EJCs. In this normal
situation, the stop codon is in the last exon and once the ribosome reaches it, there
are no more EJCs on that mRNA molecule because they have all been displaced. The
mRNA will be released in the cytosol and the protein will be translated from that
Abnormal splicing: there is a premature stop codon and it shifts the frame of the
mRNA so that the ribosome will encounter a stop codon before it is actually
supposed to. The ribosome will again bind it, go through the “test round”, but as it is
moving along and displacing EJCs, when it reaches the premature stop codon, there
will still be EJCs present in the mRNA molecule. This presence of EJCs serves as a
sign to the cell to degrade that mRNA molecule. This degradation is assisted by Upf
proteins that bind to the EJC and degrade that mRNA molecule.
This is actually a very important process in eukaryotes and is likely to have
contributed to the evolution of novel genes in eukaryotic organisms by allowing the
selection of DNA rearrangements/ alternative splice patterns that produce full
length proteins while also protecting the organism by premature stop codons that
may occur during these rearrangements.
In the immune system, there is a lot of rearrangement going on to produce all of
the antibodies involved in recognizing a diverse array of pathogens. This means
that many times the mRNA molecule produced from the rearrangements will
encode premature stop codons. In these cells, the decay will protect the cell
from many mRNA transcripts that encode premature stop codons.
About 1/3 of inherited genetic diseases are caused by premature stop codons
and the cells can degrade the aberrant mRNA to allow functional protein to
- Quality control of mRNA IN PROKARYOTES:
This is for incomplete or broken mRNAs.
If the ribosome comes to an incomplete or broken mRNA, it will stall on that molecule.
When a ribosome stalls on a prokaryotic mRNA molecule, a special type of RNA will bind to
the A-site, and this is called a tmRNA. It carries an alanine amino acid with it and as its name
implies, it acts as both a tRNA and an mRNA.
Once the tmRNA binds to the ribosome, the broken mRNA will be released from the
The tmRNA will transfer that alanine onto the growing polypeptide chain; this is an
aberrant protein because the original mRNA is damaged. This transferring of the alanine
makes the tmRNA behave as a tRNA. NOTE: there is no codon encoding for this alanine;
the tmRNA doesn’t require a codon on the corresponding mRNA to do this.
The tmRNA encodes for 10 amino acids, thereby acting as an mRNA. These 10 amino
acids will be added onto the growing polypeptide chain to create an 11-amino acid tag
(alanine + 10 amino acids encoded by the 10 codons of the tmRNA). This serves as a signal to proteases to degrade the whole protein. This 11-amino acid sequence that
makes up this tag varies for each bacteria cell but they all signal the proteases to come
and degrade the protein.
- mRNA stability:
In prokaryotes, mRNA molecules are very short-lived – less than a minute! In prokaryotes,
there are very effective exonucleases that rapidly degrade mRNA molecules. Exonucleases
degrade nucleotides/molecules from the ends and they are differentiated from
endonucleases that degrade from the inside of the molecule.
In eukaryotes, the mRNAs are more stable and can live for a maximum of 10 hours, with the
average being less than 30 minutes (in terms of stability).
There are 2 main mechanisms to degrade mRNA molecules in eukaryotes and they both
involve the gradual poly-A shortening. Recall that a properly processed mRNA molecule
has a poly-A tail at the 3’ end; once that poly-A tail is produced, it starts to slowly
degrade in the cytosol. There are about 200 A nucleotides that are added in the poly-A
tail and this tail is slowly being shortened by an exonuclease found in the cytosol called
deadenylase. This exonuclease acts as timer for the mRNA lifetime.