“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 VI. hnRNP F RRM2 Prevents RNA structure Our DNA defines who we are, encoding for proteins that carry out pre-mRNA Bases in RNA absorb UV light. UV absorption of free RNA or RNA in specific functions in our cells. Human DNA encodes for over 100,000 Exon Intron When RNA is folded up or the presence of individual RRMs proteins, and yet, recent studies have shown that humans have only 5’5’ 1 2 3 4 5 3’ structured, the bases are approximately 25,000 protein-encoding genes. Research has shown that ‘hidden’ and therefore less UV a single gene can produce multiple protein products through a process light is absorbed (black called alternative splicing. Splicing affects over 90 percent of our genome RNA is spliced spectrum in the figure to the allowing humans to be as intricate as we are. The process of alternative right). When RNA unfolds, the splicing is highly regulated by a group of splicing proteins called absorption levels increase. The 5’ 1 2 3 4 5 3’ 5’5’ 1 2 3 5 3’ heteronuclear ribonucleoproteins (hnRNPs); hnRNP F is such a protein. UV measurements confirm that hnRNP F binds RNA in a unique way compared to other hnRNPs, and new the G-folded structure is not research suggests that too much hnRNP H, a protein with a similar mRNA’s are translated into proteins present (magenta spectrum) structure and function, can manifest as a certain type of brain cancer. when hnRNP F qRRM2 is bound Understanding how hnRNP F and H bind RNA and function in alternative to the RNA, as demonstrated by Helix Helix Domnguez, et al. Structural basis of G-tract recognition and encaging by hnRNP splicing could lead to potential therapies for disease control. an increase in absorption. F quasi-RRMs. Nature Structural & Molecular Biology, vol. 17 no. 7, July 2010 Present Absent II. Gene Expression VII. Biological Significance Protein 1 Protein 2 3’ T A G C G T C C C 5’ DNA In alternative splicing, precursor messenger RNA is processed to Alternative splicing allows more than one protein to be made from one gene, as shown above. The spliceosome produce many different messenger RNAs. The expression of these removes introns and the remaining exons are ligated to form mRNA. Exons are the segments of RNA used to make Transcription different RNAs from one gene makes possible the enormous protein mRNA while introns are often ‘junk’ RNA that gets degraded. Two different mRNAs result in two different proteins and diversity found in humans. Alternative splicing affects over 90 percent this contributes to protein diversity. In this stylized example, Protein 1 has a helix contributed by exon 4, where as G C G G G of our genome, allowing humans to be as complex as we are. hnRNP F is 5’ A U C A 3’ RNA Protein 2 does not. The proteins, made from the same gene, would be functionally distinct. a protein that binds to tri-guanine sequences in pre-mRNA and Translation facilitates alternative splicing. This protein has a RRM (RNA recognition motif) that provides the first example of binding specific RNA guanines V. hnRNP F Recognizes Guanine (G) Tract Sequences in pre-mRNA (Gs) through amino acids in three loops. Classical RRMs bind RNA Ile Ala Gly Protein! through their beta-sheet surfaces. New research also suggests that over and Influences the Spliceosomes Alternative Splicing Decisions expression of hnRNP H, a protein with a similar structure and function, Proteins are made when DNA is transcribed and RNA is translated. can manifest as a certain type of brain cancer. Understanding how hnRNP F – RRM2 Model hnRNP F and H bind RNA and function in alternative splicing could lead 1 2 3 to potential therapies for disease. III. The Spliceosome Splicing No hnRNP F Enormous protein diversity results from alternative splicing. Mammals RNPs are much more complex than nematodes and fruit flies (as shown Intron 1 3 Exon 2 Skipped below), yet the genomes of these organisms differ by less than 2 fold Exon 5’ss 3’ss Exon (about 25,000, 20,000 and 14,000 genes, respectively). The extent to RNA guanine bases have the ability to hydrogen bond with which alternative splicing contributes to the complexity of eukaryotic themselves and other RNA bases, forming a folded and stable organisms is a question that remains unanswered, but a strong RNA structure (curved Gs in figure above) that is detrimental to correlation exists between complexity and intron number and splicing. Exon 2 is ‘shielded’, thus exon 2 is not included. alternative splicing. One fact that remains clear though, is that Based on alternative splicing is important for generating protein diversity. 2KG0.pdb hnRNP F Spliceosome Spliceosome GGG Disassembled Classical RRMs (RNA recognition motifs) 1 2 3 bind RNA through their beta-sheet surface Intron Removed (purple), whereas the RRM2 of the splicing with hnRNP F factor hnRNP F (above) provides the first voodoochilli.net ncbi.nlm.nih.gov house-flies.net Exons ligated, mRNA released example of binding to specific guanines (Gs) 1 2 3 Exon 2 Included primarily through amino acids in three Splicing is performed by a molecular machine called the spliceosome. hnRNP F seems to prevent the detrimental folding/structure Refrences: loops. The guanines are stacked and •Busch, A. et al. Evolution of SR protein and hnRNP splicing regulatory factors. WIREs RNA 2012, The spliceosome recognizes 5' and 3' splice sites (ss), removes introns from sequestered between hnRNP F residues (in of RNA caused by G tracts (linear Gs in figure above) allowing 3:1–12. doi: 10.1002/wrna.100 2012. the spliceosome to recognize the splice site present at exon 2, •Domnguez, et al. Structural basis of G-tract recognition and encaging by hnRNP F quasi-RRMs. pre-mRNA, and joins exons together to form the mature mRNA. mRNA is yellow) that form a ‘guanine trap’ around Nature Structural & Molecular Biology, vol. 17 no. 7, July 2010 then translated to form a protein. the Gs (light green). and so this exon is included within the mRNA. •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). .