7.5 Alternative Splicing in ammals 139Hence, multiple isoforms with structural differences are produced and regu-
lated during muscle development and adaptation. Changes in splicing enable
transitions from large to small, from acidic to basic isoforms during muscle
development [57]. Furthermore, the resulting protein isoforms show differences
in their sensitivity to Ca2+ activation and their cooperativity of contraction
[58–62]. Recently, differential expression patterns of tnnt3 pre-mRNAs were
observed in rat skeletal muscle in response to variation in body weight and also
in C2C12 muscle cells upon mechanical stretching [63, 64].
Apart from physiological adaptations, aberrant splicing of the tnnt3 gene may
contribute to disease development. An aberrant splicing pattern was identified
in myotonic diseases type 1 and 2 [65]. In mice overexpressing FRG1 (FSHD
region gene 1), aberrant splicing of the tnnt3 pre-mRNA leads to an anomalous
fast skeletal troponin T isoform that characterizes dystrophic symptoms [66].
7.5.2 Auxiliary Regulatory Elements
To allow for a correct decision as to which exon is removed or included, addi-
tional RNA sequence elements and regulatory proteins are required. A genome-
wide study of alternative splicing in mammalian tissues revealed an important
role of RNA-binding proteins in splicing regulation via their interaction with
cis-acting regulatory elements [67]. The relevant RNA sequence elements are
categorized depending on their function and position. Sequences enhancing the
splicing reaction are known as exonic splicing enhancer (ESE) or intronic splicing
enhancer (ISE), while sequences that inhibit splicing are called exonic splicing
silencer (ESS) or intronic splicing silencer (ISS) [9].
In general, splicing regulators appear to exhibit position-dependent effects on
splicing outcomes [68–70]. One family of RNA-binding proteins are the SR-rich
proteins. To interact with the RNA, they contain one or two N-terminal RNA
recognition motifs (RRMs). Additionally, they contain a unique, variable-length
RS domain at their carboxyl-terminus that functions as a protein interaction
domain [71–73]. The core SR protein family consists of 12 members, named
serine/arginine-rich splicing factors SRSF1-SRSF12, respectively [74]. SRSF1 and
SRSF2 were discovered for their essential roles in constitutive and alternative
splicing [75]. They promote both U1 snRNP binding to the 5′ splice site and U2
snRNP binding to the 3′ splice site, allowing for communication between these
recognition events [76–79], facilitating exon definition (see Section 7.2.4). The
role of SR proteins in splice site selection is discussed in Section 7.5.3. In addition,
individual SR protein expression is subject to extensive auto- and cross regulation
[80, 81]. They also interact with chromatin [82], couple with the transcription
machinery [83, 84], and are involved in mRNA export [85]. The regulation of SR
protein activity occurs at the posttranslational level. Site- or region-specific phos-
phorylation, catalyzed by specific SR protein kinases, is essential to modulate
their functions during different stages of RNA processing (reviewed in [86]).
Another family of splicing regulators is the extended family of heterogeneous
nuclear ribonucleoproteins (hnRNPs). This family includes an initially identified
set of more than 20 polypeptides, designated hnRNP A to U [87]. Their number
has further increased as many splicing isoforms, paralogs, and newly identified