Biochemistry 3382A Lecture 11: Summary Notes for Final
14 Dec 2016
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mRNA TURNOVER AND miRNA
Various mechanisms of gene regulation are coordinated together to result in an overall effect on gene
expression (histone modifications, transcription factors, post-transcriptional modifications, RNAi, etc.)
Differences between eukaryotes and prokaryotes:
- Bateia does ot hae a uleus ad so the dot euie a post-transcriptional modifications
- Bacteria have operons (polycistronic messages arranged as multiple ORFs within one transcript).
These genes usually encode proteins involved in one pathway and so operons are away for the
bacteria to regulate one pathway in a single transcription event
- Eukaotes dot hae opeos the tasipts ae ooistoi ad elated gees a be
encoded in different chromosomes). So how is one pathway regulated? Using enhancers and
silencers, RNA-binding proteins, siRNA and miRNA, mRNA turnover rates are regulated by
controlling the length of the poly-A tail, etc.
Life-Cycle of an mRNA
Both cis- and trans-
acting factors impact
mRNA half lives.
Specific sequences
located in both the
coding & non-coding
regions of different
RNAs are necessary to
trigger rapid decay
under various
conditions. Both cis-
and trans-elements are
involved in speeding or
slowing the rate of
deadenylation and cap
removal
The figure shows more control points of mRNA abundance. Trans-acting proteins are proteins from
gees eoded i a diffeet loatio, fo thei poit of effet. The oed aea aks a deisio
making poit hee the ell deides whether or not to proceed with mRNA export for translation, or to
sed the ito a gaage a fo degadatio ad tuoe, o hethe to stoe the i P-bodies
mRNA turnover encompasses all the elements of mRNA stability and decay mechanisms, and it regulates
the availability of mRNA for translation which impacts protein synthesis. It is defined as the length of
time during which an mRNA molecule is stable inside the cell. Many factors contribute to keeping the
mRNA transcript stable once it is exported to the cytoplasm (i.e. long poly-A tails) Majority of mRNAs are
unstable due to the presence of destabilizing cis-elements. These cis-elements are protected by
stabilizing factors called RNA-binding proteins (RBPs) – RBPs stabilize the cis-elements by preventing
exonuclease degradation. Normally, exonucleases are acting on the poly-A tail continuously, shortening
it over time; eventually it would reach the cis-elements and degrade it if not for RBPs
*It Is easier to regulate gene expression if you start off with an unstable mRNA
=> If majority of the mRNAs had trans-elements (making them stable), it is inefficient to add
destabilizing factors; instead degradation is an easier mechanism of stopping something
=> So it is better to start with an unstable mRNA which can be left as it is in case of inhibition
=> Stabilizing products (i.e. RBPs) can be added for subsequent stabilization when necessary
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mRNA turnover is important because:
- Functions as ‘NA ualit otol i
the nucleus after transcription to mature
mRNA (poly-A tail added and introns
spliced), but before export to cytoplasm
for translation
Why is the turnover occuring before the
mRNA is exported to the cytoplasm?
Because the cytoplasmic environment favours mRNA degradation, it will be difficult to repair any
incorrectly synthesized ‘NA tasipt ad the poess ill e less egulated ot uh of
a turnover, but rather exonucleic degradation). Also, once in the cytoplasm, the mRNA is
immediately bound by ribosomes for translation. At this point, it will be more difficult to turnover
the mRNA, since you would have to first stop translation from occuring and prevent ribosome
binding. The other disadvantage is that if turonver occurred in the cytoplasm, it would be more
difficult to recycle the nucleotides which would have to be transported back into the nucleus
- Eliminates mRNAs with mutations that could lead to synthesis of toxic proteins
Ex. Nonsense-mediated mRNA decay (NMD): translation-coupled mechanism that exists to reduce
errors in mRNA transcripts. It eliminates mRNAs containing premature translation stop codons
- Majority of mRNAs undergo decay when they are no longer needed
- Coordinating and controlling mRNA turnover is critical because change in mRNA levels can be
detrimental to the cell (Textbook pg Deeper Look
How is Half-Life of an mRNA Determined?
- It is the time at which 50% of the message remains upon the inhibition Pol-II mediated transcription.
- This technique measures the decay of mRNA over time after transcription inhibitioN
- Chemicals can be used to achieve this
Ex: Actinomycin D, α-amanitin or 5,6-dicholoro-1-β-ribobenzimidazole
SIDE NOTE: RNAP I replaces primers during replication, RNAP II transcribes protein-coding genes, RNAP III transcribes tRNA
Textbook pg. 1053 Deeper Look
The figure is being referred to in context of measuring the effect of RBP binding on mRNA stability. The
same methodology would be used to determine the half-life of an mRNA
Location of mRNA Turnover
mRNA decay occurs in a specific cellular foci called mRNA processing bodies (P-bodies). They are
cytoplamic foci consisting of RNA-protein complexes or dynamic aggregates of messenger
ribonicleoproteins (mRNPs) and P-body components. The size of P-bodies is largely dependent on the
number of non-translating mRNAs (Fig. 31.3). Under stressed conditions, there is more mRNA storage,
and thus more P-bodies in the cytoplasm
Polysome is an actively translating,
normal mRNA. When stressed, the
mRNA is recruited to the P-body
to prevent it from accessing
translation machinery. It is either
degraded by exonuclease Xrn1p,
or maybe stored for later use
mRNA DECAY MECHANISMS
How is mRNA decay carried out? After transcription in the nucleus and export to the cytoplasm for
translation, the life-cycle of normal mRNA ends with degradation mediated by cellular enzymes
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There are three pathways in which this can occur and they differ in the steps of deadenylation,
decapping, and nucleolytic degradation.
1. Deadenylation-dependent mRNA decay
2. Deadenylation-independent mRNA decay
3. Endonuclease-mediated mRNA decay
SIDE NOTE: Endonucleases cleave in the middle of a strand. Exonucleases degrade from the ends
Note that the exonuclease-mediated process (c) results in two strands, each without the post-
transcriptional modification at one end – oe stad has a fee ed hih udegoes → 5 dea;
the othe stad has a fee 5 ed ad so does ot euie deappig poteis to udego 5 → dea
Deadenylation: (Figure 31.5) A mature mRNA contains a poly-A tail between 150-200 nts. It allows for
interaction between the mRNA and the poly-A binding protein (PABP). PABP with eIF-4G circularize the
‘NA ad oes ithi age of the 5 ap, alloig the ed ad the 5 ed to iteat. This
circularization is to prevent degradation and to make translation by ribosomes more efficient (once
round one of translation is over, the ribosome can dissociate off the end and easily re-initiate translation
since the start site is right after the 3 ed. The first step in mRNA decay is shortening of the poly-A tail,
which opens up the circularized mRNA – this is mediated by deadenylase (reduces the length of the
poly-A tail to ~80 nts, and then to a critical length of 20-25 nts)
Upon deadenylation, the deadenylation-dependent mRNA decay can occur on one of two ways:
5 → dea (recruits decapping enzymes)
Decapping enzymes are to atalze the eoal of the 5 ‘NA ap because regula 5 →
eouleases aot eogize the 5 → 5 tiphosphate that makes up the methylation cap. Thus,
now that the ‘NA is eposed to ellula 5 → eoiouleases, it is rapidly degraded
*“oe iruses eode a protei alled the ap sathig protei hih atiel searhes for the hosts ‘NA ap,
and displaces it into the viral mRNA – this allows the viral mRNA to bypass exonuclease degradation and be translated
→ 5 dea (decapping enzyme-independent)
Once deadenylation is completed, the poly-A tail is shortened to its critical length. Therefore, the
ed is ustale eough to e ated upo the → 5 eoiouleases. These specific nucleases
are found within a multi-enzyme complex called the exosome (also has RNA binding activity)
Tetook pg 8 Deeper Look
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Document Summary
Various mechanisms of gene regulation are coordinated together to result in an overall effect on gene expression (histone modifications, transcription factors, post-transcriptional modifications, rnai, etc. ) Ba(cid:272)te(cid:396)ia does (cid:374)ot ha(cid:448)e a (cid:374)u(cid:272)leus a(cid:374)d so the(cid:455) do(cid:374)(cid:859)t (cid:396)e(cid:395)ui(cid:396)e a(cid:374)(cid:455) post-transcriptional modifications. Bacteria have operons (polycistronic messages arranged as multiple orfs within one transcript). These genes usually encode proteins involved in one pathway and so operons are away for the bacteria to regulate one pathway in a single transcription event. Euka(cid:396)(cid:455)otes do(cid:374)(cid:859)t ha(cid:448)e ope(cid:396)o(cid:374)s (cid:894)the t(cid:396)a(cid:374)s(cid:272)(cid:396)ipts a(cid:396)e (cid:373)o(cid:374)o(cid:272)ist(cid:396)o(cid:374)i(cid:272) a(cid:374)d (cid:396)elated ge(cid:374)es (cid:272)a(cid:374) be encoded in different chromosomes). Using enhancers and silencers, rna-binding proteins, sirna and mirna, mrna turnover rates are regulated by controlling the length of the poly-a tail, etc. Both cis- and trans- acting factors impact mrna half lives. Specific sequences located in both the coding & non-coding regions of different. Rnas are necessary to trigger rapid decay under various conditions.
