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Role in Intron Recognition

Alia Edington, Andrew MacRae, and Melissa Jurica
MCD Biology Dept, UC Santa Cruz
Splicing is a Dynamic Process 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, U3, U4, U5, and U6. The spliceosome cuts out non-coding regions of a mature
RNA, conventionally refered 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 bewteen 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 occured.

Preliminary Data: Results: Conclusion:

In vitro splicing supports alternative branch Denaturing acrylamide gel of in vitro splicing reactions
and 3 splice site usage with competitive branch sequences Preliminary data shows that our in vitro
splicing experiment supports competative
MJ76 MJ78 MJ79
Controls Strong/Strong Weak/Strong Strong/Weak branch use.
SSA (nM) Substrate
As of now, my results are less obvious
than the preliminary data tested.

Future Directions:
Repeat the experiment until the results are
Lane 1 2 3 4 5 1:30:00 mins
This gel was ran using the splicing inhibitor drug, Sequence predominent band in gel to
spliceostatin A (SSA), that has been known to interact determine their percise identity using PCR
with SF3B1 and inhibits spliceosome assembly. MJ74 is a
The results of this gel are inconclusive. The substrates containing strong
or primer extension.
pre-mRNA with a decoy branch point while MJ75 utilizes
a known substrates canonical branch point. An unknown decoy branch points (lane 3 & 5) are not significantly different to the
(star) band formed in all substrates except when the banding pattern of lanes containing weak decoy sequences (lane 1). Test this with various splicing inhibitors:
concentration elevated to 1000nM. Additionally, MJ76 and Unexpectedly, the substrate that contained a weak decoy and very strong SSA, PB, and HB
MJ78 contained an extra band indicating the use of the canonical branch point (lane 4) contained extra bands suggesting
decoy branch point. MJ79 lacked this band (boxed). misplicing 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.

Materials and Methods:

Time course of the aforementioned in vitro reaction
Canonical BP Decoy BP
Strength Strength
MJ76 Strong Strong (Con./Dec.)
Time (min) 0 4 10 45 0 4 -- 10 45 0 4 10 45 0 4 10 45 --
MJ77 Very Strong Weak
MJ78 Weak Strong
MJ79 Strong Weak
MJ80 Weak Weak

In vitro transcription and splicing Dr. Melissa Jurica
Andrew MacRae
Beth Prichard
Dr. Tonio Schtze
Veronica Urabe
John Kim
1:30:00 mins Yewande Alabi
Matt Modena