Page 62 - Academic Press Encyclopedia of Physical Science and Technology 3rd Molecular Biology

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Encyclopedia of Physical Science and Technology EN006H-655 June 29, 2001 21:21

Gene Expression, Regulation of 511

recognized through a short base pairing between the U1 splicing events for which they are required. Thus, only
snRNA and the precursor-RNA. Similarly, a base pairing around 12 “true” SR proteins have been identified. Even
between U2 snRNA and the branch point defines the 3 more surprising, gene knockout experiments suggest that
splice site. Later during spliceosome formation the U5– only one of the SR proteins is essential in Caenorhabditis
U4/U6 triple snRNP is recruited. In the triple snRNP, elegans. Thus, disrupting the expression of the SR protein
U4 and U6 snRNP form an extensive base pairing. The ASF/SF2 resulted in early embryonic lethality, whereas
catalytically active spliceosome is generated by confor- gene knockout of other SR proteins resulted in no change
mational changes, which results in a breakage of the base in phenotype. Probably, SR proteins show a large extent
pairing between U4 and U6 snRNP and formation of new of functional redundancy, and disruption of one is com-
U–UsnRNAandUsnRNA–precursor-RNAbasepairings. pensated for by another SR protein. The essential role of
It is generally believed, although not proven, that the U SR proteins in spliceosome assembly makes them prime
snRNAs in the spliceosome are the enzymes that catalyze targets for regulation of gene expression.
the two transesterification reactions required to excise the
intron.
3. The Exon Definition Model

The conserved sequences at the 5 and 3 ends of the in-
2. Non-snRNP Proteins Required for Splicing
tron are surprisingly short considering the precision by
The spliceosome, which is a large RNA–protein complex, which very large introns are excised during splicing. The
withasizesimilartoacytoplasmicribosome,alsocontains answer to this puzzle appears to be resolved by the fact

numerous non-snRNP proteins which are important for that the 5 and 3 splice sites that are joined in the splicing
correct splice site recognition. Assembly of the spliceo- reaction are not recognized over the intron. Instead splice
some proceeds over several stable intermediates (Fig. 7). sites are recognized across the exons—the so-called exon

Efficient recruitment of U2 and U1 snRNP to the 3 and definition model. Thus, whereas introns can vary in length

5 splice sites also requires specific proteins. Here only from less than 100 to more than 1 million nucleotides, in
two factors will be described. The first is U2 snRNP aux- ternal exons in a precursor-RNA have a constant length
iliary factor (U2AF), which binds to the pyrimidine tract and rarely exceed 350 nucleotides. The exon definition

located between the branch site and the 3 splice site in model postulates that U2 snRNP binding to a 3 splice site
the precursor-RNA. U2AF stabilizes U2 snRNP binding makes contact with U1 snRNP binding to the downstream

to the branch site. The second factor is not one protein, but 5 splice site (Fig. 8). If the 3 and 5 splice sites are too far
a family of proteins, designated SR proteins. SR proteins away the model postulates that the intervening sequence
contain one or two amino-terminal RNA-binding domains is not recognized as an exon because U2 and U1 snRNP
and a carboxy-terminus rich in arginine (R) and serine (S) binding to respective splice sites cannot interact with each
dipeptide repeats (the RS domain); hence the name SR other. Once the exons have been defined in the precursor-
proteins. Mechanistically, SR proteins appear to perform RNA, adjacent exons are aligned for the splicing reaction.
the same function in RNA splicing that transcriptional
enhancer proteins do in transcription initiation. Thus, SR
B. Alternative RNA Splicing Is an Important
proteins bind to splicing enhancer sequences through their
Mechanism to Generate Protein Diversity
RNA-binding domains and stimulate spliceosome assem-
bly by facilitating protein–protein interaction (Fig. 7). A major difference in gene regulation between a prokary-
The RS domain functions as a protein interaction surface otic and a eukaryotic cell is the existence of mechanisms
that makes contact with other SR proteins and so-called in eukaryotic cells that permit one gene to express mul-
SR-related proteins. Thus, many proteins involved in RNA tiple gene products. In bacteria a protein is encoded by
splicingcontainRSdomains.Forexample,SRproteinsaid a collinear DNA sequence. In contrast, in eukaryotes a

in efficient U1 snRNP binding to a 5 splice site by inter- single gene may encode for thousands of proteins. Thus,
acting with the U1-70K protein, which is an RS-domain the discontinuous arrangement of eukaryotic genes, with
containing protein. However, in contrast to transcriptional introns interrupting the coding segments of the precursor-
enhancer proteins, which active transcription irrespective RNA, permit production of multiple, alternatively spliced
of the position where they bind, SR protein function is po- mRNAs from a single gene. Examples of how a precursor-
sition dependent. Thus, in general, SR proteins function RNA can be alternatively spliced are shown in Fig. 9. This,
as splicing-enhancer proteins if they bind to the exon and of course, means that multiple proteins with different pri-
function as splicing-repressor proteins if they bind to the mary amino acid sequence and biological activity can be
intron in the precursor-RNA. produced from a single eukaryotic gene. Of specific inter-
The number of SR proteins found in mammalian cells est is that the production of alternatively spliced mRNAs
is surprisingly few considering the multitude of regulated in many cases is a regulated process, either in a temporal,