Summary Considerable information about the process of pre- mRNA splicing has accumulated, but the mechanism by which highly accurate splicing is achieved is unresolved. Fifteen years ago we proposed that accuracy in splicing might depend on small RNA molecules (splicer RNAs) which hybridise across adjacent exon termini, or intron termini. Gene expression, including alternative splicing, could be controlled by the transcription of specific splicer RNA genes. We re-assess our model here, in the light of subsequent developments.

Introduction Split genes were discovered in 1977, first in adenovirus and soon after in globin and ovalbumin Until that time it had been assumed that the DNA sequences of genes were colinear with the messenger RNA and polypeptide sequences. The discovery of exons and introns was a new challenge at various levels. Did introns have functional sig- nificance, or were they merely junk DNA? In the evolution of genes, were introns originally present, and subsequently lost in bacteria and other microbial species, or did they appear later during the evolution of eukaryotes? How were the exons spliced to form the mRNA? In subsequent years much has been learned about the mechanism of splicing. The RNA of the introns of primary transcript can be autocatalytic, and bring about self splicing (Group I and Group I1 introns) (reviewed in ref. 5). In higher organisms, splicing is usually carried out by a complex structure, the spliceosome, which consists of a number of proteins and small nuclear RNAs(~-~) . It also became apparent that there were short consensus sequences at exon-intron boundaries, as well as a consensus sequence at the branch point within the intron. In this article we only discuss splicing in higher eukaryotes.

Splicing accuracy One problem which is not yet solved is the accuracy of splic- ing. It was apparent from the beginning that to preserve the triplet coding sequence of all the exons, splicing had to be very precise. If one or two nucleotides were deleted or added during splicing, then the normal reading frame of triplets in mRNA would be disrupted. This became a formidable prob- lem when genes with many introns and exons were discov-

ered. The human dystrophin gene has at least 65 exons('O). If accuracy in splicing was 95%, then with 64 introns fewer than 1% of transcripts would produce a normal message; if accuracy was 99% then about 50% of messages would have the normal sequence. For 90% efficiency in mRNA produc- tion, the accuracy of splicing would have to be about 99.9%, or one error in 1000 splicing events. In a recent detailed review of the spliceosome mechanism of splicing in Bio-

the problem of accuracy was not considered. Our purpose in writing this paper is to stimulate discussion con- cerning the process by which accurate splicing is achieved. Thus it is hoped that researchers will consider the problem of accuracy when evaluating experiments on the mechanism of splicing. In this paper the various mechanisms for splicing are discussed with regard to the accuracy of the process. It is concluded that a mechanism based on guide RNAs is, at present, the only credible detailed mechanism by which accurate splicing can be achieved.

The consensus sequences around the splice junctions - AG/GURAGU at the 5' and (Y)nYAG/G at the 3' mam- malian splice junctions - could provide some specificity. However, sequences conforming to this consensus sequence occur elsewhere in the pre-mRNA and are not substrates for splicing. This is true even for relatively short introns, but the problem of splice-site selection becomes acute for introns of 100 kb or more, where there are thousands of possible splice sites to choose from. Thus other information is necessary to achieve accurate splicing.

One possibility is that the RNA adopts a particular confor- mation at the splice junction and, in combination with the consensus sequence, provides the specificity. No RNA sec- ondary structure has been consistently found near the splice junction. It still remains possible that a particular RNA con- formation exists around a splice site but is undetected by present algorithms. However, stable three dimensional RNA structures are usually founded on base pairing (e.g. tRNA) and since this base pairing has not been detected, this possi- bility seems unlikely. Proteins might recognise a sequence of RNA, a conformation of RNA, or both. Their binding to this structure would 'mark' the RNA to be spliced. However, the same problems apply to proteins recognising a consensus sequence andor an RNA secondary structure, as mentioned above. This protein-recognition mechanism implies that a very subtle signal is being recognised. Another problem is the very large number of proteins which would be required to achieve accuracy and specificity, especially when alternative splicing occurs (see below).

The splicer RNA hypothesis In 1979 we proposed a model to explain the high accuracy in splicing(11,12). In effect, it was derived from the related prob- lem of DNA recombination. It had long been known that when DNA molecules recombine, nucleotides are not added to or deleted from the product. This accuracy can be achieved by complementary base pairing, which guides the subse- quent breakage and reunion of polynucleotide chains(I3). Our model also depended on complementary base pairing. We proposed that small RNAs, which we called splicer RNA, are

lntron

i AoH lntrons ~ / .

\ \ Exon 1 \ Exon 2

Splicer RNA

Lariat Formation 3- 0 Branch Point

I , , . . , .

\ \

Exon 1 \ Exon 2 Splicer RNA

Fig. 1. The splicer RNA hybridises to exons 1 and 2. A ribose 2’-OH from an adenosine residue in the intron forms a covalent linkage with the 5‘-end of the intron to produce the ‘lariat’ structure. At this stage, exon 1 is not covalently linked to the rest of the RNA but is kept in place by hybridisation to the splicer RNA. The splicer RNA, which has brought the ends of exon 1 and exon 2 into close proxim- ity, then facilitates exon joining. The figure shows splicer RNA hybridised to exon sequences, but alternative structures could occur. Splicer RNA could hybridise to intron sequences instead of exon sequences. Alternatively, splicer RNAs could hybridise to both exon and intron sequences. A further variation could use the specificity of hybridisation to allow accurate joining during ‘lariat’ formation. By hybridising to the appropriate sections of the intron, a splicer RNA could bring the ribose 2’-OH from an adenosine residue in the intron into close proximity with the exon I-intron boundary to allow accurate ‘lariat’ formation to take place.

transcribed, and these exactly hybridise to the termini of adjacent exon sequences, or alternatively the termini of introns, or possibly both (Fig. 1). This structure would guide the subsequent breakage and reunion of the single RNA polynucleotide chain, so that adjacent coding sequences were exactly contiguous. The SnRNP particle, which was subse- quently discovered, could provide the active machinery to bring about this process. It would always act at the 5‘ and 3’ exon sites which splicer RNA had made contiguous, irre- spective of the actual nucleotide sequences surrounding the junction. According to this model, there would be a large number of splicer RNAs, each capable of hybridising to the appropriate transcript sequences. SnRNPs, like ribosomes, would not be specific; the specificity of their action would depend on other informational RNA molecules. This also meant that the small nuclear RNA could be characterised because they are common to all (or most) SnRNPs, whereas the splicer RNAs we invoked would be heterogeneous, and

\ Antisense Pseudogene Transcript

Fig. 2. The accurate splicing of several exon sequences may be guided by an antisense transcript from a processed pseudogene, which lacks introns. Provided accurate hybridisation occurs across exon termini, some imperfect matching of bases could occur within exons.

therefore much harder to detect. However, there need not be a different splicer RNA for each splice site. It is possible that an antisense sequence to several exons could be responsible for the removal of several introns. Nevertheless, our model is based on the supposition that there are very large numbers of DNA sequences coding for antisense RNAs, which hybridise to adjacent exon sequences in pre-mRNAs, or alternatively to the termini of intron sequences, or both. Alternatively, it is possible that sequences reside within introns, which have the same ability to guide accurate splicing as splicer RNA tran- scribed elsewhere in the genome. The existence of such sequences would be a special case of the general model, which proposes that intron RNA has a catalytic role in splic- ing. It could be tested by scrutiny of long introns to determine whether antisense sequences exist which are complementary to exonhntron junctions in the same transcript.

Fig. 2 illustrates the possibility that processed pseudo- genes might have a role in splicing. It is commonly believed that these pseudogenes arise from the DNA copy of a mRNA which is subsequently inserted into the chromosome. They therefore have no introns, and because they are inactive genes, they can mutate to produce sequences which no longer have the original open reading frame (ORF). It is now known that at least some ‘pseudogenes’ are transcribed either from the sense strand or from the opposite strand to produce anti- sense m ~ l e c u l e s ( ’ ~ ~ ’ ~ ) . It is possible that antisense transcripts have a role in splicing, as shown in Fig. 2, and it is important to note that this could be achieved even though there are some nucleotide differences between the exon sequences and the pseudogene transcript.

Alternative splicing In our 1979 papers, we proposed that split genes provide an opportunity for the regulation of gene activity during devel- opment, or in different tissues. This would depend on the presence or absence of particular splicer RNAs, which would allow the normal processing of some RNA transcripts and not others. In subsequent years, more and more examples of alternative splicing have been uncovered. In some cases 15- 20 alternative mRNAs arise from a single gene(I6.I7). The exons can be divided into constitutive sequences which are

present in all mRNAs, and exon sequences which are present in some mRNAs and not others. The combinational rules of alternative splicing can be complex; for example, in the fast skeletal troponin T gene, five consecutive exons are spliced in a combinatorial fashion to yield 32 different sequences in the protein, and these are bounded by constant exon sequences(17). As Smith et a1.(I6) have explained, alternative splicing provides a means of producing families of proteins with related function, and these are referred to as isoforms. Thus, the specific characteristics of a protein can be varied according to developmental stage, specific cell type or the physiological condition of the cell. The evolution of split genes provides a mechanism whereby many proteins are pro- duced from one gene, which is a more efficient mechanism than, for example, gene duplication and divergence.

It is usually believed that alternative splicing is controlled by proteins(6g18), but our hypothesis provides a more econom- ical mechanism. It could be the regulation of splicer RNA synthesis that determines whether a particular pattern of splicing will occur in a particular situation (Fig. 3). If alterna- tive splicing depended on proteins, a very large number would presumably be required to control the synthesis of a large family of isoforms.

Is there any experimental evidence that could exclude our proposal or any of the other possible mechanisms for splic- ing? Experiments have been carried out to duplicate splice j u n c t i o n ~ ( l ~ > ~ ~ ) . However, the interpretation of these results, which have been mainly carried out with cell-free extracts, has not allowed a definite judgement to be made. Recently, the construction of hairpin structures, which include the con- sensus sequences, has shown that splicing is strongly inhib- ited(20). Such hairpins would prevent the normal hybridisa- tion of splicer RNA (Fig. 1).

Problems with the splicer RNA hypothesis There are several potential problems with the splicer RNA hypothesis. Firstly, it proposes that there are a large number of genes coding for splicer RNAs, but there is, as yet, an apparent lack of genetic evidence for these genes. As men- tioned above, splicer RNAs could be coded in antisense pseudogenes, or possibly SnRNA pseudogenes. Splicer RNA genes would be small, and there could be multiple copies; more importantly, mutants of splicer RNA genes might not be recognised as such by investigators.

The second problem concerns the strong evidence that SnRNAs can base pair with introns and exons at the position where the splicer RNA hybridised9). The interactions with SnRNAs are weak, involving 1 to 6 base pairs, which are not always complementary. The SnRNA pairing is mainly with intron sequences, leaving the exons free to hybridise with splicer RNA (see Figs 1-3). In addition we believe the anal- ogy to genetic recombination is valid, with splicer RNA pro- viding strong specific interaction, just as with hybrid DNA; the spliceosome then acts on this specific junction to break and rejoin the RNA molecule, just as the recombination pro- teins act on hybrid DNA to break and rejoin the DNA.

The third problem concerns the discovery of proteins involved in alternative splicing, e.g. tra and tra-2 and sex

lntron 2

Exonl \ ) Exon 2 \ ,,/ Exon3

Splicer RNA 1 Splicer RNA 2

Exon 2

Splicer RNA 3

Fig. 3. Alternate splicing of exons could be achieved by different splicer RNAs. Two such RNAs are needed to accurately splice together exons 1 , 2 and 3 (top). In the absence of these splicer RNAs, a different molecule could bring about the joining of exons 1 and 3 (bottom).

selection in Drosophila('8,28). Our model does not exclude the possibility that accessory proteins could be required for alternative splicing, but we postulate that a splicer RNA pro- vides the specificity. (It is also possible that the regulation of pre-mRNA splicing in sex determination is a special case, as it is controlled by an auto-regulatory loop.)

Testing the splicer RNA hypothesis There are several ways to test the splicer RNA hypothesis. A search could be made for genes that are homologous to splice junctions. Erythroid cells could be used to search for fairly abundant small RNAs that are homologous to the exon, or intron, termini of globin genes. Sequence databases could be examined for intron or other sequences that are homologous to the exon / intron termini (such as the large HPRT gene, the beta globin cluster, and so on).

Experiments utilising heterologous splicing could be a powerful means of determining the mechanism of accurate splicing. When a gene containing introns is transcribed in a cell from a different species, splicing can still take place. For example, Drosophila genes are spliced in human extracts or Xenopus oocytes('8,21 1. This suggests that intron sequences might play a crucial role in the splicing reaction. However, most of the experiments have been carried out with cell-free extracts and it is uncertain whether the products seen are accurately spliced. To determine complete accuracy it would be necessary to use primers hybridising to adjacent exons and

then to isolate and sequence the exon-exon boundary follow- ing PCR (polymerase chain reaction). It is hard to see how an accurate splicing mechanism could be universal and com- posed of simple elements found in all or many organisms. Also, when the transacting factors required in alternative splicing are considered, the problem becomes more complex. If alternative splicing occurs in different tissues in one organ- ism, would the factors responsible be present in other organ- isms, which may not carry out alternative splicing? This is a problem that applies to the control of splicing, whether by protein factors or by splicer RNAs. Clearly the relationships between alternative splicing and heterologous splicing need careful examination, as well as the accuracy of splicing.

Significance of splicing Soon after the discovery of split genes, it was proposed that exons represent primitive mini-genes, usually with short cod- ing sequences(22). During evolution these coding sequences could be re-assorted to produce proteins with different domains coded for by different exons. This exon-shuffling hypothesis proposes that introns are of ancient origin, that in present-day microbial species have usually been lost from genes(23). In contrast, the ‘intron-late’ theory of gene evolu- tion proposes that a mechanism evolved whereby one gene can produce a family of related proteins(16). It is important to note that the ‘intron-early’ theory of gene evolution has a direct parallel in the alternative splicing seen in present-day higher organisms. Alternative splicing can bring together dif- ferent polypeptide domains into one protein. Some parts of the protein (constitutive exons) are essential for function, but others have a more specific role, related perhaps to interaction with other proteins, with nucleic acids or with an allosteric response to a small effector. In other words, alternative splic- ing provides the means of juggling conserved polypeptide domains.

If small splicer RNAs really exist, it is possible that they could move from cell to cell. It is remarkable that mam- malian cells will not take up single nucleotides, nor high mol- ecular weight DNA or RNA, but it is well known that oligonucleotides containing 15-30 bases will cross the mem- brane(24). There is evidence for receptors which facilitate uptake of these m ~ l e c u l e d ~ ~ ~ ~ ~ ) . So this could be a normal process for transfer of information from cell to cell. More specifically, the transfer of splicer RNAs could control the particular mode of splicing which will occur in a target cell. The possibility of RNA-mediated cell-cell signalling opens up many intriguing possibilities in development.

The functions of RNA molecules are remarkably diverse: it is the message which mediates the transfer of information from DNA to protein; RNA is an integral component of ribo- somes and spliceosomes; it has catalytic properties, as seen in the self-splicing of Group I and Group I1 introns and in ribozymes; it is the adaptor which makes the specific connec- tion between an amino acid and its cognate triplet in the mes- sage; it can be the normal genome of RNA viruses, and it is widely believed it was the crucial molecule in the origin of life. It has also been suggested it might have a morphogenetic role in cells in early development(27). We propose here that

RNA also controls the accuracy of pre-mRNA splicing by sequence-specific complementary base pairing.

Acknowledgment We thank Jonathan Izant for helpful discussions.

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Robin Holliday is at the CSIRO Division of Biomolecular Engineering, Sydney Laboratory, PO Box 184, North Ryde, NSW 21 13, Australia, and Vincent Murray is at the School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, NSW 2052, Australia.