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In Vitro Studies of Self-Splicing Group II Introns

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In vitro studies of self-splicing group II introns

Hebbar, Sharda Kattingeri, Ph.D.

The Ohio State University, 1989

Copyright ©1989 by Hebbar, Sharda Kattingeri. Allrightsreserved.

U-M-I

300N.ZecbRd. Ann Arbor, MI48106

IN VITRO STUDIES OF SELF-SPLICING GROUP II INTRONS

DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate
School of the Ohio State University

By
Sharda Kattingeri Hebbar, B.S., M.S.

* * * * *

The Ohio State University
1989

Dissertation Committee: P. A. Fuerst
Approved by
L. F. Johnson

—

A. M. Lambowitz P. S. Perlman
* Adviser
Department of Molecular Genetics
Copyright by
Sharda Kattingeri Hebbar
1989

To My Parents, and To My Husband, Raja.

ACKNOWLEDGEMENTS

I would like to thank Dr. P.S. Perlman for his assistance and guidance. Special thanks go to Kevin Jarrell and Rosemary Dietrich not only for their technical advice but also for their friendship. To my husband, Raja, I would like to express my sincere gratitude for his encouragement, support and endurance throughout my endeavors.

iii
VITA

February 21, 1980
1960 ............. Born - Andhra Pradesh, India
................... B«S., Osnania University,
Hyderabad, India

1980 - 1982

1983 - 1986
................... M.S., Andhra University,
Waltair, Andhra Pradesh

.................... Graduate Teaching Associate,
MCDB Program, The Ohio State University, Columbus, Ohio

1986 - 1988

.................

.

Graduate Research Associate, Department of Molecular Genetics, The Ohio Sta+e University, Columbus, Ohio

FIELDS OF STUDY
Major Field: Molecular Genetics

TABLE OF CONTENTS

INTRODUCTION .................................................

1
I.A. Catalytic RNA I.B. Yeast mitochondrialDNA
I.B.l. Mosaic genes

..........................

.

........

12235

6

...............................

I.B.2. Maturases I.B.3. Group I introns
I.B.3.a. Discovery of RNA catalysis I.B.3.b. Tetrahymena rRNA intron self-splices by a two step transesterification pathway
I.B.3.C. Tetrahymena rRNA intron is a group I intron
79
I.B.3.d. Mitochondrial group I introns self-splice in vitro

10

I.B.3.e. Not all group I introns are self- splicing

11

I.B.3.f. The intervening sequence RNA of
Tetrahymena is an Enzyme
I.B.3.g. Active site model
12 13
I.B.4. Group II intron splicing ...................... 14
I.B.4.a. Conserved sequence elements and secondary structure ........................... 14
I.B.4.b. Group II introns in yeast

mitochondria ................................... 16

I.B.4.C. Group II introns encode proteins related to reverse transcriptase..................................,,17
I.B.4.d. AI5g self-splices in vitro by a

novel..........................

18
I.B.4.e. Role of branch formation ................ 19 I.B.4.f. Group II intron splicing resembles nuclear pre-mRNA splicing .......... 20
I.B.4.g. Alternative reaction conditions

.......

22

I.B.4.h. Trans-splicing .......................... 23 I.B.4.i. Dependance on 5’ exon

sequences...............................

25
I.B.4.j. Multiple 5’exon binding sites .......... 26 I.B.4.k. Domain 4 is dispensible ................. 26 I.B.4.1. Domain 5 is required for 5’

exon release....................................27

I.C. Dissertation Goals.........
28

v
29

II.A. Plasmid constructions ............................... 29

MATERIALS AND METHODS

II.B. Plasmid preparation............... 30 II.C. Transcription and Purification of RNA..............30 II.D. All Splicing reactions............ II.E. Site-directed mutagenesis.......... II.F. Purification and end-labeling of
30 31

oligonucleotides ....................................... 31

II.G. RNA sequencing..................................,....32 II.H. Northern Blots....................................... 32

  • II.I. 3* end-labeling of RNA.......................
  • 32

II.J. Limit T1 digest...................................... 32 U.K. Other Methods........................................ 33 II.L. Enzyme reagents........
33

RESULTS...........................................

34
III.A. Test of the generality of group II self-

splicing ..............................

34
III.A.I. Cloning of intron 1 of the COX I gene

............................................. 35

III.B. Features of the all self-splicing

reaction ................................. 36

.......

*
III.B.1. All RNA is inactive in the standard aI5g splicing buffer............................... 36
III.B.2. All has an absolute requirement for monovalent cations.................
III.B.3. All RNA has a higher threshold for

»........37

magnesium than aI5g................................ 38
III.B.4. NH4C1 as the standard splicing

buffer..................................

III.B.5. All reactions have a temperature optimum of 4QoC............................
III.B.6 . The reaction is unimolecular and time dependent . .....
39 40 40
III.B.7. Optimum reaction conditions ................ 41
III.C. Characterization of the NH4C1 reaction products 42
III.C.l. Identification of products containing the 3*exon...........
III.C.2. First test of the validity of the

assignments...................

43 44
IiI.C.3. Accurate ligation of exons ................. 45 III.C.4. The slowly migrating RNA species is excised intron lariat (IVS-LAR) .................. 46
III.C.5. Accurate 5’ Cleavage ........................ 47 III.C.6 . Technical problems in mapping the

branch point ....................................... 48

III.C.7. Summary and implications of product characterization

.............................48

vi

III.D. All yields some novel reactionproducts in KC1
III.D.1. Further analysis of KC1 effects on
49 the all reaction

............................. 50

III.D.2. Characterization of novel KC1 products

..........................51

III.D.3 A proposed pathway for the KC1 reaction.......
54

55 55 57 58
III.E. Spliced exon reopening (SER)
III.E.l. All does not reopen spliced exons......... III.E.2. SER is sequence specific...................
III.F. Much of the intron ORF can bedeleted
III.F.l. Location of the branch site in the

  • shortened IVS-LAR..........
  • -

...........

60 62 62
I1I.G. Studies using portions of all
III.G.1. Trans-splicing

.................

III.G.2. The conserved 5*boundary sequence is needed for trans-splicing......
III.H. Summary

.

...........

.

64

66

III.I. Studies of aI5g self-splicing
111.1.1. Introduction...........

  • -
  • 67

67
111.1.2. Domain 5 is required, in cis, for

the second step of splicing........................68

111.1.3. Mutational analysis of the conserved 5* end of the intron

.......

71
111.1.3.a. The first 7 nt of the intron are essential for branch formation............ 71
111.1.3.b. 5’ end of the intron plays a role in SER.....................................75
111.1.4. Domain 6 is not required for splicing in vitro..........
76

  • 77
  • III.J. Molecular dissection of domain 5

III.J.l. Introduction................................. 77 III.J.2. Heterologous experiments...... III.J.3. Mutations of the highly conserved unpaired regions of domain 5
77

...... 78

III.J.3.a. The 4 base pair loop................. 78 III.J.3.b. RNA truncated at the Rsa I site in domain 5 is inactive.................. 80
III.J.3.0. The CG bulge...........................81
III.J.4. Bottom helix ................................. 83 III.J.5. 7 nt in the 5* half of domain 5 can form a perfect match with 7nt in domain 1

.................................................... 85

DISCUSSION

88

IV.A. All self-splices in vitro

88

IV.A.l. All self-splices under conditions different from those of aI5g and bll .............90
IV.A.2. All reaction pathways ........................ 91 IV.A.3. All undergoes a post-splicing

vii

reaction in KC1.....

........................

IV.A.4. All does not carry out apliced exon reopening

.

........

.

.......................

.

IV.A.5. Much of the intron open reading

frame can be deleted.......................

IV.B. 5* end of the intron is not required for 5' exon release but plays a role in branch formation
IV.C.. 5* end of the intron plays a role in SER IV.D. Lariat formation is not required for efficient splicing in vitro
.. 93
94 96

96 97
IV.E. Domain 5 is also required for the second step in splicing
IV.E.l. 4 base GAAA loop is not critical for

domain 5 function ...........................

IV.E.2. The 2 base bulge is crucial for domain 5 function

.

...........................

100
IV.F. Future experiments
IV.F.l. Point of interaction..................... IV.F.2. Transformation experiments

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