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The SR Protein 9G8 and the Wilms' Tumor Suppressor Protein WTI

Promote Translation of mRNAs with Retained Introns

Jennifer Elizabeth Swartz

Vermontville, Michigan

B.S., Western Michigan University, 1995

A Dissertation presented to the Graduate Faculty

of the University of Virginia in Candidacy for the Degree of

Doctor of Philosophy

Department of Microbiology

University of Virginia

December 2007

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? Copyright by

Jennifer Swartz

All Rights Reserved

January 2008

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Abstract

Recent data indicate that the various steps in post-transcriptional gene expression are linked. In the studies

presented in this thesis, we show further evidence of the interconnection between the multiple steps of

mRNA metabolism. In one study, we show a link between splicing with nucleocytoplasmic export and

translation. In another study, we show that the products of an alternatively spliced pre-mRNA have

different roles in mRNA metabolism. Specifically, SR proteins, Tap, and WT1(+KTS) function similarly to

link nuclear and cytoplasmic events in the expression of mRNAs with retained introns.

The first study examines the cytoplasmic role of the splicing regulatory SR protein, 9G8, which

has recently been proposed to function in mRNA export in conjunction with the export protein, Tap/NXF1.

We investigated the effect of 9G8 on cytoplasmic RNA fate. 9G8 was shown to enhance expression of

unspliced RNA containing either the MPMV-CTE or the recently discovered Tap-CTE. 9G8 also enhanced

polyribosome association of unspliced RNA containing a CTE. Hyperphosphorylated 9G8 was present in

monosomes and small polyribosomes, whereas soluble fractions contained only hypophosphorylated

protein. Our results are consistent with a model in which hypophosphorylated SR proteins remain stably

associated with mRNP complexes during export and are released during translation initiation concomitant

with increased phosphorylation.

The second study examines the Wilms¡¯ tumor 1 (WT1) gene, which plays an important role in

mammalian urogenital development. Dysregulation of this gene is observed in many human cancers.

Alternative splicing of WT1 RNA leads to the expression of two major protein isoforms, WT1(+KTS) and

WT1(-KTS). Whereas WT1(-KTS) acts as a transcriptional regulator, no clear function has been ascribed

to WT1(+KTS), despite the fact that this protein is crucial for normal development. Here we show that

WT1(+KTS) functions to enhance expression from RNA possessing a retained intron and containing either

a cellular or viral constitutive transport element (CTE). WT1(+KTS) expression increases the levels of

unspliced RNA containing a CTE and specifically promotes the association of this RNA with

polyribosomes. These studies provide further support for links between different steps in RNA metabolism

and for the existence of post-transcriptional operons.

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Acknowledgements

I foremost thank Doctors Lou Hammarskj?ld and David Rekosh for their support, both financial and

intellectual, during my graduate training at the University of Virginia. Their great knowledge of Virology,

molecular biology techniques, and the historical perspectives have been invaluable. I respect greatly their

scientific ethics and their insistence on doing what they thought was right, regardless of any pressure to

compromise from their peers. I thank the members of my committee, Coleen McNamara, Lucy Pemberton,

and Margo Roberts, who gave me nothing but support and encouragement, even when the project was not

going well. Lucy, in particular, who not only joined the committee late in the process, but who eventually

became my primary reader, was extremely helpful in producing the final dissertation document. My

colleagues in the lab have been friends, mentors, and helpful assistants. From the lab techs (Joy, Sue, Ryan,

Dawn, Fei Fei) who helped make the lab run so smoothly, to the post docs (Bernhard, Ian, Lionel, Yeoucherng, Jin, Ying, Yuming, Lucia, John, Yukiko, and Sharmila) who shared their vast experience, to the

students (Tiffany, Brian, Kate, Melissa, Greg, Kevin, Alex, Deidre) who learned beside me and

commiserated with me, I have enjoyed getting to know and work with each of them. In particular, Yeoucherng has been like another mentor to me, giving me support beyond just technical knowledge and

assistance. Not only did he help me with my research in performing the Northern Blots for all of my

experiments, he worked closely with me helping me to think about experiment design and how confusing

results could be interpreted. He helped me countless times trouble-shooting problems with the gradient

fractionator or just keeping me company while processing gradients. He also provided me with emotional

support whether it was through philosophical discussion or quiet example. I will forever be grateful for

Yeou-cherng¡¯s interest in my professional and personal development. Gloria has also played a great role in

keeping me sane. She was always there to comfort me or help me negotiate the university labyrinth.

Everyone knows how important Martha had been to the graduate students in the department, and one of my

biggest disappointments was not finishing my graduate work before she retired. Luckily, Sandy has done a

brilliant job stepping into her shoes, and I am glad I got to know her, as well. In addition to Lou, David and

Margo, I also thank the other members of the Thaler Center, Dean Kedes and David Camerini and the

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members of their labs. Dean, in particular as been a good friend, and I greatly appreciate the interest and

support he has given me. Jay Brown and Bill Newcomb have also been great friends to me, and I¡¯ve

enjoyed the time I¡¯ve spent with them. Jack Schell has been such a good friend, as well, and this really

intensified over the last several years as our graduate careers took very similar paths. I think a difficult time

was made much more tolerable for each of us by having someone to commiserate with that understood the

other¡¯s experience so fully. I also thank my family, who were proud of me even though they didn¡¯t

understand a thing of what I said when I talked about my work, and who didn¡¯t nag me about when I¡¯d be

done. That kind of support is invaluable. I thank my judo community who let me take out my frustrations

on them and then thanked me with a smile after a vigorous workout. Finally, I thank Victor, who came into

my life right at the end, and although he knew nothing about the many years leading up to this point, he

gave me so much support and motivation that the process became a joy rather than a painful exercise.

Because of him, I will remember the writing process with fondness. I couldn¡¯t ask for a better way to

conclude my graduate career.

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