The spliceosome is a dynamic molecular machine found in the nucleus of cells that

has a critical function in gene expression. The spliceosome is composed of small

nuclear ribonucleoproteins (snRNPs) complexes made from five U-rich snRNAs: U1,
U2, U4, U5, and U6. The spliceosome cuts out non-coding regions of a mature RNA,
conventionally referred to as introns, and ligates the remaining coding regions
known as exons. The newly spliced product is further translated to produce proteins
for a multitude of functions for the cell.

The purpose of this project is to understand the role of the U2 snRNP splicing
protein, SF3B1, in intron recognition. SF3B1 will be tested against different
substrates in its ability discriminate between a "decoy" branch point of alternating
strength placed upstream of the normal branch point. The spliced products will then
be analyzed by their size to determine if proper recognition has occurred.

MJ76 Denaturing acrylamide gel of in vitro splicing reactions Preliminary data shows that our in vitro
MJ78 MJ79
Controls Strong/Strong Weak/Strong Strong/Weak
with competitive branch sequences splicing experiment supports competitive
branch use.
SSA (nM)
00

00

00
0

0
10

10

10
10

10

10
10

10

10
0

0
1

As of now, my results are less obvious

M 4
5
J7
J7
M

Substrate than the preliminary data tested.

(Con./Dec.)

*
This gel was ran using the splicing inhibitor drug,
spliceostatin A (SSA), that has been known to interact
with SF3B1 to change its conformation from that required
for regulation of spliceosome assembly. MJ74 originates
from a cDNA for a known substrate with a decoy branch
point while MJ75 comes from a cDNA utilizing the
canonical branch point of a known substrate. An unknown
(star) band formed in all substrates except when the
concentration elevated to 1000nM. Additionally, MJ76 and
MJ78 contained an extra band indicating the use of the
decoy branch point. MJ79 lacked this band (boxed).
Repeat the experiment until the results are
conclusive.
30W Sequence predominant band in gel to
Lane 1 2 3 4 5
1:30:00 mins
determine their precise identity using PCR
or primer extension.
The results of this gel were inconclusive to the preliminary data. The
substrates containing strong decoy branch points (lane 3 & 5) were not Test this with various splicing inhibitors:
significantly different to the banding pattern of lanes containing weak decoy SSA, PB, and HB
sequences (lane 1). Unexpectedly, the substrate that contained a weak
decoy and very strong canonical branch point (lane 4) contained extra
bands suggesting missplicing had occurred. The substrate in lane 2 was
lost due to residual ethanol. No controls were used in this experiment,
therefore, band identification was speculated.

Canonical BP Decoy BP
Time course of the aforementioned in vitro reaction
Substrates
Strength Strength
Substrate
MJ76 Strong Strong S/S VS/W W/S S/W
(Con./Dec.)

MJ77 Very Strong Weak Time (min) 0 4 10 45 0 4 -- 10 45 0 4 10 45 0 4 10 45 --

MJ78 Weak Strong

MJ79 Strong Weak
MJ80 Weak Weak

In vitro transcription Dr. Melissa Jurica

Andrew MacRae
Beth Prichard
Dr. Tonio Schtze
Veronica Urabe
John Kim
30W
1:30:00 mins Yewande Alabi
Matt Modena