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“The Guanine Trap”
RNA Guanine-tract Recognition and Encaging by hnRNP F quasi-RRM2
Valders High School SMART Team: Grace L. Ebert, Theresa M. Evenson, Elizabeth F. Evans, Phoenix J.M. Kaufmann, Nicole C. Maala,
Mitchel R. Meissen, Paige N. Neumeyer, Alexis A. Patynski, Ian M. Schmidt
Instructor: Mr. Joseph S. Kinscher Mentor: Mark T. McNally, Ph.D., Medical College of Wisconsin
2011-2012 Valders S.M.A.R.T. Team
Abstract: RNA splicing, the process where mRNA exons are ligated together after the introns are cut out, is required for the production of mature mRNA. Exons are the regions of mRNA that are translated into protein, and introns are noncoding regions. Alternative splicing, the process
where different combinations of exons can be ligated together to generate many mRNAs from one gene, accounts for protein diversity and affects over 90 percent of human genes. Alternative splicing regulation is important because many diseases can arise if it occurs improperly. A molecular
machine called the spliceosome performs RNA splicing to ligate exons, and hnRNP F (heterogeneous nuclear ribonucleoprotein) influences the spliceosome's alternative splicing decisions. hnRNP F binds to guanine-(G) rich sequences in mRNA targets, resulting in their alternative splicing.
Modeling shows a 'cage' formed around three G residues, which explains why hnRNP F binds G-rich sequences. Using 3D printing technology, the Valders SMART team (Students Modeling A Research Topic) modeled hnRNP F binding via arginine 116, phenylalanine 120, and tyrosine 180.
Research suggests that too much hnRNP H, a close homologue of hnRNP F, plays a role in promoting brain cancer. Understanding how hnRNP F binds G-rich RNA to cause alternative splicing may lead to the development of therapies for genetic diseases.
I. Introduction
IV. Alternative Splicing
Our DNA defines who we are, encoding for proteins that carry out
specific functions in our cells. Human DNA encodes for over 100,000
proteins, and yet, recent studies have shown that humans have only
approximately 25,000 protein-encoding genes. Research has shown that
a single gene can produce multiple protein products through a process
called alternative splicing. Splicing affects over 90 percent of our genome
allowing humans to be as intricate as we are. The process of alternative
splicing is highly regulated by a group of splicing proteins called
heteronuclear ribonucleoproteins (hnRNPs); hnRNP F is such a protein.
hnRNP F binds RNA in a unique way compared to other hnRNPs, and new
research suggests that too much hnRNP H, a protein with a similar
structure and function, can manifest as a certain type of brain cancer.
Understanding how hnRNP F and H bind RNA and function in alternative
splicing could lead to potential therapies for disease control.
Exon
pre-mRNA
Intron
1
5’
VI. hnRNP F RRM2 Prevents RNA structure
2
3
5
4
3’
RNA is spliced
5’
1
2
3
4
5
3’
5’
5’
1
2
3
5
3’
mRNA’s are translated into proteins
Helix
Absent
Helix
Present
Bases in RNA absorb UV light.
When RNA is folded up or
structured, the bases are
‘hidden’ and therefore less UV
light is absorbed (black
spectrum in the figure to the
right). When RNA unfolds, the
absorption levels increase. The
UV measurements confirm that
the G-folded structure is not
present (magenta spectrum)
when hnRNP F qRRM2 is bound
to the RNA, as demonstrated by
an increase in absorption.
II. Gene Expression
C G T
3’ T A G
G C A
C C C 5’
DNA
G G G 3’
RNA
Protein 1
Protein 2
Alternative splicing allows more than one protein to be made from one gene, as shown above. The spliceosome
removes introns and the remaining exons are ligated to form mRNA. Exons are the segments of RNA used to make
mRNA while introns are often ‘junk’ RNA that gets degraded. Two different mRNAs result in two different proteins and
this contributes to protein diversity. In this stylized example, Protein 1 has a helix contributed by exon 4, where as
Protein 2 does not. The proteins, made from the same gene, would be functionally distinct.
Translation
Ile
Ala
Gly
Protein!
Proteins are made when DNA is transcribed and RNA is translated.
V. hnRNP F Recognizes Guanine (G) Tract Sequences in pre-mRNA
and Influences the Spliceosomes Alternative Splicing Decisions
hnRNP F – RRM2 Model
III. The Spliceosome
1
Splicing
RNPs
2
Spliceosome
Disassembled
3
No hnRNP F
1
Intron
Exon 5’ss
Domnguez, et al. Structural basis of G-tract recognition and encaging by hnRNP
F quasi-RRMs. Nature Structural & Molecular Biology, vol. 17 no. 7, July 2010
VII. Biological Significance
Transcription
5’ A U C
UV absorption of free RNA or RNA in
the presence of individual RRMs
3’ss Exon
3
Exon 2 Skipped
RNA guanine bases have the ability to hydrogen bond with
themselves and other RNA bases, forming a folded and stable
RNA structure (curved Gs in figure above) that is detrimental to
splicing. Exon 2 is ‘shielded’, thus exon 2 is not included.
Based on
2KG0.pdb
Spliceosome
Intron Removed
Exons ligated, mRNA released
Splicing is performed by a molecular machine called the spliceosome.
The spliceosome recognizes 5' and 3' splice sites (ss), removes introns from
pre-mRNA, and joins exons together to form the mature mRNA. mRNA is
then translated to form a protein.
Classical RRMs (RNA recognition motifs)
bind RNA through their beta-sheet surface
(purple), whereas the RRM2 of the splicing
factor hnRNP F (above) provides the first
example of binding to specific guanines (Gs)
primarily through amino acids in three
loops. The guanines are stacked and
sequestered between hnRNP F residues (in
yellow) that form a ‘guanine trap’ around
the Gs (light green).
In alternative splicing, precursor messenger RNA is processed to
produce many different messenger RNAs. The expression of these
different RNAs from one gene makes possible the enormous protein
diversity found in humans. Alternative splicing affects over 90 percent
of our genome, allowing humans to be as complex as we are. hnRNP F is
a protein that binds to tri-guanine sequences in pre-mRNA and
facilitates alternative splicing. This protein has a RRM (RNA recognition
motif) that provides the first example of binding specific RNA guanines
(Gs) through amino acids in three loops. Classical RRMs bind RNA
through their beta-sheet surfaces. New research also suggests that over
expression of hnRNP H, a protein with a similar structure and function,
can manifest as a certain type of brain cancer. Understanding how
hnRNP F and H bind RNA and function in alternative splicing could lead
to potential therapies for disease.
Enormous protein diversity results from alternative splicing. Mammals
are much more complex than nematodes and fruit flies (as shown
below), yet the genomes of these organisms differ by less than 2 fold
(about 25,000, 20,000 and 14,000 genes, respectively). The extent to
which alternative splicing contributes to the complexity of eukaryotic
organisms is a question that remains unanswered, but a strong
correlation exists between complexity and intron number and
alternative splicing. One fact that remains clear though, is that
alternative splicing is important for generating protein diversity.
hnRNP F
1
2
GGG
3
with hnRNP F
1
2
3
Exon 2 Included
hnRNP F seems to prevent the detrimental folding/structure
of RNA caused by G tracts (linear Gs in figure above) allowing
the spliceosome to recognize the splice site present at exon 2,
and so this exon is included within the mRNA.
voodoochilli.net
ncbi.nlm.nih.gov
house-flies.net
Refrences:
•Busch, A. et al. Evolution of SR protein and hnRNP splicing regulatory factors. WIREs RNA 2012,
3:1–12. doi: 10.1002/wrna.100 2012.
•Domnguez, et al. Structural basis of G-tract recognition and encaging by hnRNP F quasi-RRMs.
Nature Structural & Molecular Biology, vol. 17 no. 7, July 2010
•Nilsen, T. et al. Expansion of the eukaryotic proteome by alternative splicing. Nature, vol. 463,
January 2010
A SMART Team project supported by the National Institutes of Health Science Education Partnership Award (NIH-SEPA 1R25RR022749) and an NIH CTSA Award (UL1RR031973).