MS-1 FUND 1: 9:00-10:00
Transcriber: Aditi Jani
Tuesday, September 23, 2014
Editor: Wilson DeLaney
Dr. Ching-Yi Chen
Post-Transcriptional Regulation Part II
Page 1 of 3
Introductory Comments: This is the second part of Dr. Chen’s lecture on Post-Transcriptional Regulation. Time stamps
correspond to ECHO recording.
I.
snRNPs Form the Spliceosome (slide 39) 0:10 SN: Included key points from first slide, as Dr. Chen was finishing up the answer to a
student question during the break that was not on the ECHO recording.
a. Splicing occurs when the snRNPs come together with the pre-mRNA to form a complex called the spliceosome.
The assembly of this complex requires ATP. SN: Dr. Chen emphasized that the spliceosome complex is dynamic- the snRNP’s
join and leave the complex at particular points in the splicing process.
II.
III.
IV.
V.
VI.
VII.
b. snRNP U1 binds at the 5’ splice site, and snRNP U2 binds at the branch site on the pre-mRNA.
c. Interaction between the snRNPs brings the 5’ and 3’ splice sites together to form the lariat and to allow for exon
ligation.
d. The transesterification reactions that join the exons may be catalyzed by snRNAs themselves, but not the
snRNP proteins
e. Spliceosome assembly requires ATP-dependent RNA rearrangements catalyzed by spliceosome DEAD-box
ATPases/helicases. SN: Dr. Chen did not go into detail on this topic
The spliceosome- RNA/Protein Complex (slide 40) 0:45
a. The spliceosome contains RNA (snRNA, or small nuclear RNA).
b. There are five snRNAs- U1, U2, U4, U5, U6.
i. They are uridine-rich, hence the “U,” and they are very small (<200 nucleotides long).
ii. Each snRNA assembles with at least 10 different proteins to form snRNP’s (small nuclear
ribonucloproteins).
iii. In addition to snRNP’s, there are also proteins involved in splicing which are called non-snRNP splicing
factors.
iv. The current estimate of total proteins found in spliceosome is > 50 different proteins.
snRNPs Form the Spliceosome (slide 41) 1:47
a. The spliceosome reaction is dynamic, but the first event is when U1 interacts with the 5’ splice site
i. U1 forms a characteristic secondary structure
ii. The 5’ single stranded end of U1 snRNA will base pair with 5’ splice site, so they can interact. The same
thing occurs with U2.
Assembly of the Spliceosome (slide 42) 2:37
a. There is a recycling of each snRNA. Once the intron is spliced out, the U1, U2, etc. will be recycled to do
another round of splicing.
Constitutive Splicing vs. Alternative Splicing (slide 43) 2:59
a. Regulation occurs surrounding splicing.
b. Two kinds of splicing: constitutive splicing and alternative splicing
c. Constitutive splicing means that every exon is present in the mature mRNA (no exons are skipped). This
produces a single form of mature mRNA from the primary mRNA transcript. If every intron is removed, every
exon is joined together, and you’ll only make one mature mRNA.
d. This is not the case for many eukaryotic cells, so there is alternative splicing: the purpose is to make different
isoforms of RNA from a single gene. This can be accomplished in several ways:
i. Using different promoters
ii. Selection of different polyadenylation sites (sometimes there will be a few of them, and you can select a
different one and create a different isoform)
iii. Alternative splicing of the primary transcript
iv. A combination of these three mechanisms
Constitutive Splicing (slide 44) 5:31
a. Constitutive splicing: every intron is removed and incorporated into the mature mRNA, without exception.
b. Slide shows “A cell” and “B cell,” indicating that constitutive splicing occurs in both cell types.
c. Only one mRNA isoform is produced from the same gene in both A and B cells.
Alternative mRNA Splicing (slide 45-48) 6:16-12:05
a. Alternative splicing, one or a few exons will be skipped in the mature mRNA. An exon can be present or it may
not be.
b. In some cases, multiple cell types will undergo the same alternative splicing
i. Example from slide: A cell and B cell have the same isoform of mRNA
c. In other cases, alternative splicing depends on the cell type.
i. Example from slide: A cell has exon II, B cell has two different isoforms of the mRNA, and in the C cell,
exon II is always skipped
MS-1 FUND 1: 9:00-10:00
Transcriber: Aditi Jani
Tuesday, September 23, 2014
Editor: Wilson DeLaney
Dr. Ching-Yi Chen
Post-Transcriptional Regulation Part II
Page 2 of 3
d. Alternative splicing is the mechanism that can create protein isoforms (slide 47) 8:45
i. Example of fast skeletal troponin gene in skeletal muscle
ii. Contains 18 exons; 11 of the 18 are constitutively spliced (always there) in mature RNA.
iii. 5 of the exons (#4-8) are alternatively spliced, in various combinations– either none will be there, some
may be there, or all may be present.
iv. If you calculated, there would be 32 possibilities of alternative splicing.
v. Exons 16 and 17 are mutually exclusive, that is, one is always present but never both. So there are 2
possibilities here.
vi. How many possible mature mRNA’s can be made? 64 (32 times 2)
e. Another example (slide 48) 10:54: - tropomyosin gene – contains three possible poly-A signals.
i. Depending on where the gene is expressed (cell type), there is alternative splicing and different use of
poly-A signals.
1. In striated muscle, the first poly-A signal is used
2. In smooth or fibroblast cells, the third poly-A signal is used
ii. This allows you to increase the coding capacity of the gene even beyond just alternative splicing.
VIII. mRNA Export (slide 49-50) 12:12-13:25
a. After processing, the mRNA contains 5’ cap, 3’ poly-A tail, and the introns are spliced out (absent).
b. Now the mRNA needs to be exported form the nucleus, and there is a level of regulation here called mRNA
export control. SN: He says that we are not going into detail on this
c. The RNA is synthesized and packed with different proteins, and the 5’ end gets exported first. Some of the
proteins will be exported to the cytosol along with the RNA, some will remain in nucleus.
d. In the cytosol, the CAP-binding protein will be replaced by translation initiation complex and the mRNA will be
circularized
IX. Post-Transcriptional Regulation in the Cytoplasm (slide 51) 13:25
a. There are at least three possible fates of mRNA in the cytosol:
i. Translation – this is the main purpose (SN: He mentions that this is not his topic for this lecture)
ii. Degradation – depends on the nature of the RNA. Some will be degraded more quickly than other RNA’s,
based on degradation control.
iii. Localization, then translation.
X. Degradation and mRNA stability (slide 52) 14:20
a. In prokaryotes: mRNA are very unstable. Synthesized and then degraded rapidly- this is why bacteria can grow
and respond to environmental signals very quickly.
b. Eukaryotes: more stable RNA’s with a half-life of more than 10 hours. This is due to the 5’ cap, 3’ poly-A tail,
and the RNA is circularized. All of these things help protect it from degradation and keeps it stable.
i. However there are some RNA’s with half-life shorter than 30 minutes.
1. These RNA’s encode signaling molecules or regulatory proteins (like onco genes) that need to be
expressed transiently and degraded rapidly in order to respond to environmental changes.
2. mRNA’s that encode growth factors, transcription factors, and oncogenes fit this category.
XI. Two mechanisms of Eukaryotic mRNA Decay (slide 53) 16:38
a. General decay pathway
i. This occurs in the majority of mRNA
ii. First event of degradation is removal of the poly-A tail, then poly-A binding protein can no longer bind to
the RNA and inhibit degradation.
iii. The RNA without poly-A will be degraded later either in the 5’  3’ or 3’  5’ direction.
b. The minor pathway: Certain mRNA’s require specific sequences where the cleavage occurs.
i. Cleavage occurs in the middle of the RNA sequence, making it into two shorter parts.
ii. This pathway no longer requires poly-A shortening
iii. It can also be degraded in either direction (5’  3’ or 3’  5’)
XII. RNA Editing (slide 54-56) 20:24-26:12
a. Another modification which belongs to post-transcriptional regulation
b. This is another way to increase the diversity of genetic information
c. RNA editing only changes a few (or one) nucleotides in an mRNA
i. Occurs by deaminating a base, and it’s a very rare occurrence in mammalian cells.
ii. Two main ways it occurs: Adenine  Inosine or Cytosine  Uridine
iii. These changes will alter the coding possibilities (because I will pair with C, not U as A does), and U will pair
with A (not G as C does).
d. This increases protein diversity by altering amino acid coding possibilities, introducing a premature stop codon,
or changing the splice sites in a transcript.
e. A to I editing: (slide 55) 22:28
i. Adenosine will be converted into inosine (A  I)
MS-1 FUND 1: 9:00-10:00
Transcriber: Aditi Jani
Tuesday, September 23, 2014
Editor: Wilson DeLaney
Dr. Ching-Yi Chen
Post-Transcriptional Regulation Part II
Page 3 of 3
1. Adenosine deaminase enzyme will act on the RNA, recognize the secondary structure, and remove
the amino group and it will become a hydroxyl group.
2. This enzyme is an RNA-binding protein and a deaminase
f. Editing of apolipoprotein B (apoB): C to U
i. Requires a protein complex called an editosome
1. The editosome contains a cytosine deaminase and an adaptor protein that helps in recognition and
in bringing the deaminase and the transcript together.
ii. C to U editing depends on the way apoB is expressed.
1. If the gene is expressed in the liver, the RNA will not be edited and will be translated into a full apoB
protein.
2. If in intestine, the editosome will edit the C  U, such that the CAA codon becomes UAA, which is a
stop codon. If the RNA is then translated, you will make a truncated isoform of apoB called apoB-48.
iii. Editing depends on cell type in this case
XIII.Unified Theory of Gene Expression (Slide 57-58) 26:14- 32:54
a. There are stages involving regulation of gene expression:
i. Transcription, transcript processing, mRNA export, and then translation
b. Traditionally we think that each event needs to be complete for the next event to begin (i.e.- transcription needs
to be completely finished before RNA processing can begin), but this is totally wrong!
i. The events are not independent of each other
ii. Each stage is part of a continuous process with physical and functional connections between transcriptional
and processing machinery.
c. Capping, RNA splicing, 3’ poly-A tail formation, and even nuclear export, are coupled to transcriptional
machinery
i. If you mutate RNA polymerase II or inhibit translation, you will still make mRNA, but capping will not
happen, 3’ polyadenylation will not happen, etc. (Processing will not occur properly).
d. Regulation occurs at multiple levels in this continuum.
e. Additionally, eukaryotic cells also have elaborate mRNA surveillance systems
i. This means that if for some reason the RNA is not capped, or some of the introns are not removed, the
RNA will not even have a chance to get into the cytosol – it will be degraded in the nucleus.
ii. Surveillance mechanism: if anything wrong happens in processing steps, the RNA will not leave the
nucleus and will be degraded. (SN: he says that he did not speak about the surveillance mechanism in great detail. Just know
that it exists.)
f. Processing steps coupled (SN: see image on slide 58)
i. When RNA polymerase II begins synthesis of mRNA, the capping machinery will add the 5’ cap as soon as
a few nucleotides are transcribed because the capping machinery associates with RNA pol II to begin with.
ii. If you have a mutation and it disrupts this interaction, even though RNA polymerase II can synthesize the
mRNA, the capping will not happen.
iii. Similarly, when introns are synthesized, the splicing machinery will recognize the intron and assemble onto
the intron. But, splicing may not occur immediately even though the intron has been specified– it depends –
some introns splicing can occur during transcription and some after the 3’ poly-A tail is added.
g. When the 3’ end of the mRNA is forming, the 5’ end has already been exported to the cytosol
h. mRNA in the cytosol can be translated into protein, some will be degraded faster than others, or it will be
transported to a specific cellular compartment before translation occurs (this is mRNA localization control).
i. The whole process, at least for the nuclear events, occurs simultaneously.
Student Questions
Student Question 1: Does the 5’ cap and poly-A tail not spontaneously re-circularize?
Answer: If you remove poly A, circularization can’t occur. When the RNA circularized, another protein may
somehow recognize the RNA, bind to it, and loosen the complex, so other enzymes can get access to degrade it.
Translational machinery is engaged. If you somehow inhibit translation, there is competition between translation
and mRNA degradation. If you inhibit translation, degradation will predominate.
Student Question 2: Where does RNA editing occur?
Answer: In the nucleus.
Student Question 3: Explain one more time about RNA degradation and oncogene activity?
Answer: Oncogenes need to be expressed for only short periods of time, so one way to make sure that happens
is to make less RNA, but the other way is to degrade the RNA rapidly. If you inhibited RNA degradation, the
oncogene RNA will be degraded slower than normally needed and prolong the expression of the oncogene. If the
oncogene is required is for cell growth, the cells will grow quickly. If it’s required for tumor formation, tumors will
form.
<END OF LECTURE 32:54>