1.

We discussed the association between different posttranscriptional events (PowerPoint:

Integrating Polyadenylation, Capping and RNA Polymerase).

a. Describe an experiment that indicates a relationship between either the 5’ cap and
splicing or the polyA tail and splicing.

In the 1991 Niwa and Berget paper published in Genes and Development the authors investigated
the relationship between polyadenylation and splicing of pre-mRNAs with variable numbers of
identical exons under low and high magnesium concentrations. They found that high magnesium
concentration in vitro tends to decouple the stimulating effect of polyadenylation on removal of
the intron most proximal to the poly(A) site whereas low magnesium concentration tends to
enhance the stimulating effect. Under low magnesium concentration, the authors found that
splicing of pre-mRNA containing a single intron was significantly enhanced in wild type RNA
(with poly(A) consensus sequence AAUAAA) compared to RNA containing a mutation in the
poly(A) signal (AAGAAA) that effectively stops polyadenylation from occurring. The authors
showed this by running the mRNA products on electrophoretic gel at different time intervals
during the processing reactions. The wild type pre-mRNA resulted in fully processed mRNA
(spliced and polyadenylated) and partially processed mRNA (only spliced or only
polyadenylated). A pre-mRNA version that completely lacked a poly(A) signal and contained
only splicing signals resulted in significantly lower levels of the spliced mRNA product (no
polyadenylation of course). The mutated pre-mRNA contained barely detectable levels of spliced
mRNA product with no polyadenylation whatsoever. The authors ran essentially the same
experiment on pre-mRNAs that contained two introns with identical splicing signals to show that
removal of the first intron did not depend on prior polyadenylation (kinetics of intron 1 removal
were overlapping for wt and mutated pre-mRNA) but removal of the second intron did depend
on prior polyadenylation (intron 2 removed from wt pre-mRNA more efficiently than from
mutated version). The authors showed the same pattern in a 3 intron pre-mRNA system.

b. What types of events are associated with the phosphorylated carboxyl terminal domain
of RNA polII?

The CTD of RNA polII can exist in nonphosphorylated and highly phosphorylated forms. The
CTD serves as a flexible binging scaffold for different nuclear proteins. Binding of specific
proteins is determined by the pattern of phosphorylation on the CTD repeats which serves as a
code for RNA synthesis and processing. The pattern of phosphorylation changes as RNA polII
moves across a gene and thus different proteins are recruited to the CTD at different times in
RNA synthesis. Some of the many different proteins that bind to the CTD include splicing
factors, polyadenylation factors, and 5' capping enzymes.

2. The article referenced below described the first details of how short RNAs targets and
silences mRNA.

Zamore, P.D., T. Tuschl, P.A. Sharp and D.P. Bartel. RNAi: Double-Stranded RNA
Directs the ATP-Dependent Cleavage of mRNA at 21 to 23 Nucleotide Intervals.
Cell 101:25-33
In one experiment, labeled sense and/or antisense dsRNA was used to target artificial
mRNA in Drosophila embryo lysates that carried out RNAi in vitro. After labeled dsRNA
was used in lysates, small RNAs were identified by electrophoresis.

a. In the figure representing these results, what evidence is there that targeted mRNA does
not need to be present for the production of short 21-23 nt RNAs?

Lanes 2&3, 4&5, 6&7, and 9&10 show that equal amounts of 21-23 nt RNA cleavage products
resulted from reactions containing both lysate and target mRNA as well reactions containing
only lysate (no mRNA). These lanes also show that both the sense and antisense strands in
dsRNA were cleaved equally.

Labeled luciferase mRNA was targeted using various fragments of the dsRNA. After
allowing cleavage by the RNAi machinery, cleaved mRNA fragments and their lengths
were identified using gel electrophoresis.

b. What is the relationship between cleavage site on the mRNA and the short guide RNA?
The short guide RNA fragments base pair with the mRNA and define the boundaries of cleavage
along the mRNA sequence. RNAi mediated cleavage can only occur within the base paired
domain.

c. Why are the differences between fragment sizes significant?

The differences between mRNA cleavage fragments are significant because this result strongly
suggests that RNAi mediated cleavage occurs only along regions of the mRNA that base pair
with the short guide RNA fragments.

d. Why are fragments generated with dsRNA A longer than fragments generated with
dsRNAs B and C.

Fragments generated with dsRNA A are longer than fragments generated by the two other
dsRNAs because the 5' end of dsRNA A is furthest from the 5' end of the mRNA. Since the
mRNA is 32P-labeled at its 5' cap cleavage products from the dsRNA A trigger will be longer
than cleavage products from the other two triggers.

e. Why is the use of “high resolution gel electrophoresis” necessary?

High resolution gel electrophoresis is necessary in order to count the spacing between cleavage
fragments with the use of the T1 partial base-hydrolysis ladder.

3. Distinguish between the location and functions of Dicer, Drosha and Argonaute proteins.

Drosha is an RNase III-like enzyme located in the nucleus that functions to initiate the
processing of pre-miRNA by cleaving it to produce a characteristic stem-loop structure of 65-70
base pairs in length with a two base overhang on the 3' end that is later recognized by Dicer.
Drosha forms the microprocessor complex with the dsRNA binding protein Pasha which is
required for its ribonuclease activity.

Dicer is an RNase III-like enzyme located in the cytoplasm that functions to cleave dsRNA and
pre-miRNA into siRNA, short dsRNA fragments 21-23 nucleotides in length.

Argonaute is a family of proteins located in the cytoplasm that bind the guide strand of siRNA
and form the catalytic component of the RISC complex. Ago2 is the only argonaute protein that
has endonuclease activity directed against mRNA strands with extensive complementarity to the
Ago2 bound siRNA guide strand. Ago2 performs the "slicer" activity of the RISC complex.

4. Cells regulate gene expression at various places in the process of gene expression.
Regulation of transcription allows cells to regulate before energy and resources have been
used to express an unneeded gene. Why is it also advantageous for cells to regulate at the
level of translation?

It is advantageous to regulate gene expression at the level of translation because this allows a cell
to make a rapid response to external stimuli. It's faster to modulate the rate of translation of
endogenous mRNA than it is to synthesize new mRNA and in some cases mRNA is
overproduced and stored temporarily for purposes of rapid protein synthesis when the cell needs
it immediately. Translational control also confers the benefit of fine tuning protein production
because it is one of the last steps in the whole complex process. Furthermore, translational
control adds redundancy into gene expression which is important if another step of regulation
fails.

The following paper investigates the function of let-7 repression of target mRNAs:
Pillai, R. S., S.N. Bhattacharyya, C.G. Artus, T. Zoller, N. Cougot, E. Basyuk,
E. Bertrand, and W. Filipowicz. 2005. Inhibition of Translational Initiation
by Let-7 MicroRNA in Human Cells. Science 309:1573-1576.

5. mRNA abundance is analyzed from constructs containing various constructed 3’ UTRs.

a. How do the 3’ UTR regions differ in the experimental “3XBulge” and “Con” constructs?

The "3XBulge" mRNA construct contains 3 repeats of a sequence that is partially complimentary
to that of let-7, allowing the formation of a bulged duplex with the let-7 RNA. "Con" mRNA
contains no sites that allow duplex formation with let-t RNA.

b. Why is there almost no mRNA detected when the “Perf” construct is used?

"Perf" mRNA contains a single perfectly complementary site to let-7 RNA and as a result "Perf"
mRNA is targeted by let-7 and cleaved by the RISC complex.

6. To determine whether expression is regulated at the level of translation or protein

degradation, they target their protein to the ER during expression. How do the results
support the conclusion that this gene is not regulated at the level of protein degradation?
The authors use the above Western blot to show that the tagged protein did successfully localize
to the ER. According to the protein degradation hypothesis, localization of luciferase to the ER
will protect it from degradation and therefore we should see increased luciferase activity. But
what we see instead from the reporter assay is that hAgo2 tethering-associated repression of
luciferase persists even when the protein is tagged for localization to the ER (fig. 1B). Thus
protein degradation alone cannot explain the reduced luciferase activity observed.

7. A target mRNA expressing two luciferase genes is constructed. Translation of the FL

gene is cap-dependent while translation of the RL gene is not.

a. What is the significance of the 2 BoxB region in this experiment?

The 2 BoxB hairpin region serves as a site for tethering of the translation initiation factors eIF4E
or eIF4G which drives translation of the RL cistron.

b. FL expression is inhibited when translation initiation factors are expressed with the
mRNA, but repression of RL is relieved. Explain why this is the case.

In translation initiation 4E must first bind to the 5' cap of the mRNA followed by 4G and other
factors in order to form the initiation complex. The authors propose that partial binding of let-7
to the 3xBulge may prevent recognition of the 5' cap by 4E or possibly recruitment of 4G during
initiation. Since the presence of the 2 BoxB hairpin loops allows 4E and 4G to bind to the
intercistronic region the RL cistron can be translated even when let-7 is bound to the 3xBulge.
Binding of let-7 prevents translation of the FL cistron most likely because it prevents recruitment
of 4E to the 5' cap.