ANNUAL REVIEWS Annu. Rev. Cell Bioi. 1992. 8:133-55 Copyright
©
Further
Quick links to online content
1992 by Annual Reviews 1nc. All rights reserved
Annu. Rev. Cell. Biol. 1992.8:133-155. Downloaded from www.annualreviews.org Access provided by University of California - San Francisco UCSF on 11/19/14. For personal use only.
ALTERNATIVE mRNA SPLICING Michael McKeown Molecular Biology and Virology Laboratory, The Salk Institute, P.O. Box San Diego, California
92186
85800,
KEY WORDS: RNA processing, gene regulation, mRNA splicing
CONTENTS 133
INTRODUCTION REVIEW OF THE MECHANISM OF SPLICING
... . . . . . . . . . . . ... . ... . . . . . .
KINDS OF ALTERNATIVE SPLICING AND THEORETICAL CONSIDERATIONS FOR STUDYING THEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
134 135
SPLICE/DON'T SPLICE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
RPL32 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P-Element . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . su(w") . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . transformer-2 in ehe Male Germline . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
137 137 1 38 138 139
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
141
5' OR 3' SPLICE SITE CHOICE .
.
5' Splice Site Choice--SV40 T A ntigen ................................. 3' Splice Site Choice--transformer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
142
144
. . . .
146 146 147 148
MUTUALLY EXCLUSIVE EXON USE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tropomyosin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
148 148
3' EXON CHOICE
150 150 151
EXON SKIPPING . . . . . . . . . . . . . . . Sex-lethal . . . . . . . . . . . . . . . . . . . ras . . . . . . . . . . . . . . . . . . . . . . . . Multiple Exon Skipping--CD45 . . .
. . . .
. . . .
. . . .
. . . .
. . . .
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. . . .
. . . .
. . . .
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. .. . . . . . . . . . . . . .
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... . . . . . . . . .
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... . . . . . . . . .
. . . .
. . . .
. . . .
. . . .
.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Calcironin/CGRP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . doublesex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
INTRODUCTION Alternative splicing regulates the production of a number of important biological molecules . This regulation can involve on/off regulation of the products of particular genes, or it can involve the production of alternative products with clearly separable functions. From the number of genes that are
133 0743-4634192/1115-0133$02.00
Annu. Rev. Cell. Biol. 1992.8:133-155. Downloaded from www.annualreviews.org Access provided by University of California - San Francisco UCSF on 11/19/14. For personal use only.
134
MCKEOWN
now known to be regulated by alternative splicing, it is clear that splicing regulation is a critical adjunct to the regulation of promoter activity. This review cannot cover all of the observed cases of alternative splicing. Instead, it focuses on a limited number of cases that represent the possible kinds of alternative splicing. These cases are chosen specifically because they lead to insight into the mechanisms by which alternative splicing can occur and point out certain general features and problems in the study of regulated alternative splicing. From even the small amount that we know about alternative splicing, it appears that there are multiple mechanisms that bring about splicing regulation and that each example gives insight into a particular mechanism only. Much of the work described in this review involves studies of in vivo regulation of alternative splicing. In addition, there is a bias toward systems in which it is possible, by genetic means, to identify key regulatory factors that control alternative splicing events and that establish useful in vivo systems for tests of cis-regulatory elements in the RNA. Finally, it should be noted that alternative splicing might be a much more prevalent regulatory mechanism than now realized. The cases in which we are aware of regulation by alternative processing are precisely those cases in which the RNA products of alternative processing events can be readily observed (specifically the RNAs must either be stable or identifiable from genetic considerations). If, on the other hand, a nonfunctional RNA turned over rapidly relative to an alternatively spliced translated RNA, it is possible that the nonfunctional RNA would not be noticed or, if noticed, ignored as being at too Iow a level to be relevant. Alternatively, if a functional RNA is present at low levels relative to the nonfunctional alternatively spliced molecules, we might fail to notice it and assume that the nonfunctional RNAs are the important molecules. These are not idle theoretical speculations; in fact, an example of the first possibility (ras) and two examples of the second (su(wa) and P-element) are described as part of this review.
REVIEW OF THE MECHANISM OF SPLICING Much is now known about the biochemical mechanism of splicing. It is beyond the scope of this review to cover all of that material. The reader is directed to several recent reviews for greater details (Green 1991; Ruby & Abelson 1991) . Figure 1 diagrams the outlines of what happens to the RNA during splicing and defines a number of sites on the RNA that will be important in our discussion. Note that the actual mRNA splicing reaction involves the recognition of a number of these sites by specific proteins and ribonucleoprot ein complexes. Ultimate understanding of the regulation of alternative splicing will require understanding how specific regulatory factors interact with these
ALTERNATIVE SPLICING
135
Intron -=-5' SpIice-,
Site
Branch Point
3' Splice Site
1 Exon 1 �AGjGURAGU-----YNCUGAC--YnNYAG 1 1 + 1....Ex_. o_n _---'�OH __
....--
'--__.!.!..-...J
Exon2
'-----...J polypyrimidine
Annu. Rev. Cell. Biol. 1992.8:133-155. Downloaded from www.annualreviews.org Access provided by University of California - San Francisco UCSF on 11/19/14. For personal use only.
tract
_1
1 Exon 1
Exon2
1
Q
Figure 1
The RNA processing pathway during RNA splicing.
"basal" splicing factors, but at the moment the mechanism of such interactions is not understood.
KINDS OF ALTERNATIVE SPLICING AND THEORETICAL CONSIDERATIONS FOR STUDYING THEM Figure 2 shows a schematic representation of many of the various alternative splicing events that can occur. These are arranged in approximately an increasing order of complexity, extending from simple splice/don't splice decisions, through competing 5' or 3' splice sites, single and multiple exon skipping, to mutually exclusive exon use. Finally, two cases are presented in which additional alternative non-splicing events also occur: alternative 5' ends with alternative 5' splice sites, and alternative 3' ends with alternative 3' splice sites. Note that in these last two cases, regulation could be at the level of a 5 or 3' end choice with splicing choices following from the first choice in a manner dependent only on the basal splicing machinery. In the study of each of these mechanisms of alternative splicing there are a few simplifying assumptions that can lead to the development of simple testable hypotheses. The experiments derived from these assumptions should easily indicate if additional more complicated methods of regulation are occurring. The first assumption is that in most cases only one of the splice sites is actually regulated. A corollary of this assumption is that the other sites only respond to the basal splicing machinery as it is affected by the context produced by the regulated event. The second assumption is that regulation is actually occurring in only one of the two potential expressing tissues, with
Annu. Rev. Cell. Biol. 1992.8:133-155. Downloaded from www.annualreviews.org Access provided by University of California - San Francisco UCSF on 11/19/14. For personal use only.
136
MCKEOWN
C::::: :::= �I :( (�..,,----.,
©
Further
Quick links to online content
1992 by Annual Reviews 1nc. All rights reserved
Annu. Rev. Cell. Biol. 1992.8:133-155. Downloaded from www.annualreviews.org Access provided by University of California - San Francisco UCSF on 11/19/14. For personal use only.
ALTERNATIVE mRNA SPLICING Michael McKeown Molecular Biology and Virology Laboratory, The Salk Institute, P.O. Box San Diego, California
92186
85800,
KEY WORDS: RNA processing, gene regulation, mRNA splicing
CONTENTS 133
INTRODUCTION REVIEW OF THE MECHANISM OF SPLICING
... . . . . . . . . . . . ... . ... . . . . . .
KINDS OF ALTERNATIVE SPLICING AND THEORETICAL CONSIDERATIONS FOR STUDYING THEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
134 135
SPLICE/DON'T SPLICE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
RPL32 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P-Element . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . su(w") . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . transformer-2 in ehe Male Germline . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
137 137 1 38 138 139
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
141
5' OR 3' SPLICE SITE CHOICE .
.
5' Splice Site Choice--SV40 T A ntigen ................................. 3' Splice Site Choice--transformer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
142
144
. . . .
146 146 147 148
MUTUALLY EXCLUSIVE EXON USE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tropomyosin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
148 148
3' EXON CHOICE
150 150 151
EXON SKIPPING . . . . . . . . . . . . . . . Sex-lethal . . . . . . . . . . . . . . . . . . . ras . . . . . . . . . . . . . . . . . . . . . . . . Multiple Exon Skipping--CD45 . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. . . .
. .. . . . . . . . . . . . . .
. . . .
. . . .
. . . .
. . . .
. . . .
... . . . . . . . . .
. . . .
. . . .
... . . . . . . . . .
. . . .
. . . .
. . . .
. . . .
.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Calcironin/CGRP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . doublesex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
INTRODUCTION Alternative splicing regulates the production of a number of important biological molecules . This regulation can involve on/off regulation of the products of particular genes, or it can involve the production of alternative products with clearly separable functions. From the number of genes that are
133 0743-4634192/1115-0133$02.00
Annu. Rev. Cell. Biol. 1992.8:133-155. Downloaded from www.annualreviews.org Access provided by University of California - San Francisco UCSF on 11/19/14. For personal use only.
134
MCKEOWN
now known to be regulated by alternative splicing, it is clear that splicing regulation is a critical adjunct to the regulation of promoter activity. This review cannot cover all of the observed cases of alternative splicing. Instead, it focuses on a limited number of cases that represent the possible kinds of alternative splicing. These cases are chosen specifically because they lead to insight into the mechanisms by which alternative splicing can occur and point out certain general features and problems in the study of regulated alternative splicing. From even the small amount that we know about alternative splicing, it appears that there are multiple mechanisms that bring about splicing regulation and that each example gives insight into a particular mechanism only. Much of the work described in this review involves studies of in vivo regulation of alternative splicing. In addition, there is a bias toward systems in which it is possible, by genetic means, to identify key regulatory factors that control alternative splicing events and that establish useful in vivo systems for tests of cis-regulatory elements in the RNA. Finally, it should be noted that alternative splicing might be a much more prevalent regulatory mechanism than now realized. The cases in which we are aware of regulation by alternative processing are precisely those cases in which the RNA products of alternative processing events can be readily observed (specifically the RNAs must either be stable or identifiable from genetic considerations). If, on the other hand, a nonfunctional RNA turned over rapidly relative to an alternatively spliced translated RNA, it is possible that the nonfunctional RNA would not be noticed or, if noticed, ignored as being at too Iow a level to be relevant. Alternatively, if a functional RNA is present at low levels relative to the nonfunctional alternatively spliced molecules, we might fail to notice it and assume that the nonfunctional RNAs are the important molecules. These are not idle theoretical speculations; in fact, an example of the first possibility (ras) and two examples of the second (su(wa) and P-element) are described as part of this review.
REVIEW OF THE MECHANISM OF SPLICING Much is now known about the biochemical mechanism of splicing. It is beyond the scope of this review to cover all of that material. The reader is directed to several recent reviews for greater details (Green 1991; Ruby & Abelson 1991) . Figure 1 diagrams the outlines of what happens to the RNA during splicing and defines a number of sites on the RNA that will be important in our discussion. Note that the actual mRNA splicing reaction involves the recognition of a number of these sites by specific proteins and ribonucleoprot ein complexes. Ultimate understanding of the regulation of alternative splicing will require understanding how specific regulatory factors interact with these
ALTERNATIVE SPLICING
135
Intron -=-5' SpIice-,
Site
Branch Point
3' Splice Site
1 Exon 1 �AGjGURAGU-----YNCUGAC--YnNYAG 1 1 + 1....Ex_. o_n _---'�OH __
....--
'--__.!.!..-...J
Exon2
'-----...J polypyrimidine
Annu. Rev. Cell. Biol. 1992.8:133-155. Downloaded from www.annualreviews.org Access provided by University of California - San Francisco UCSF on 11/19/14. For personal use only.
tract
_1
1 Exon 1
Exon2
1
Q
Figure 1
The RNA processing pathway during RNA splicing.
"basal" splicing factors, but at the moment the mechanism of such interactions is not understood.
KINDS OF ALTERNATIVE SPLICING AND THEORETICAL CONSIDERATIONS FOR STUDYING THEM Figure 2 shows a schematic representation of many of the various alternative splicing events that can occur. These are arranged in approximately an increasing order of complexity, extending from simple splice/don't splice decisions, through competing 5' or 3' splice sites, single and multiple exon skipping, to mutually exclusive exon use. Finally, two cases are presented in which additional alternative non-splicing events also occur: alternative 5' ends with alternative 5' splice sites, and alternative 3' ends with alternative 3' splice sites. Note that in these last two cases, regulation could be at the level of a 5 or 3' end choice with splicing choices following from the first choice in a manner dependent only on the basal splicing machinery. In the study of each of these mechanisms of alternative splicing there are a few simplifying assumptions that can lead to the development of simple testable hypotheses. The experiments derived from these assumptions should easily indicate if additional more complicated methods of regulation are occurring. The first assumption is that in most cases only one of the splice sites is actually regulated. A corollary of this assumption is that the other sites only respond to the basal splicing machinery as it is affected by the context produced by the regulated event. The second assumption is that regulation is actually occurring in only one of the two potential expressing tissues, with
Annu. Rev. Cell. Biol. 1992.8:133-155. Downloaded from www.annualreviews.org Access provided by University of California - San Francisco UCSF on 11/19/14. For personal use only.
136
MCKEOWN
C::::: :::= �I :( (�..,,----.,
