140 7 Splicing and Alternative Splicing Impact on Gene Design
proteins have been added based on structural and functional considerations
(reviewed in [88]). Affiliated are the following proteins: Nova, Sam68, QKI, TDP-
43, TIA, Hu, Fox, CUG-BP, MBNL, and ESRP proteins [88]. hnRNPs share a
modular structure, most frequently containing one or more RRMs, one of the
most abundant protein domains found in eukaryotes [89, 90]. The K homology
(KH)-type RNA-binding domain occurs in the hnRNP proteins K and E and in
the hnRNP-like proteins Nova, Sam68, and QKI [88]. In addition to that, many
hnRNPs contain RGG boxes (repeats of Arg-Gly-Gly) and other auxiliary
domains, such as acidic and glycine- or proline-rich domains [91, 92]. Most
hnRNPs shuttle between nucleus and cytoplasm [93].
A few examples shall give an overview of the multifunctionality of hnRNPs:
hnRNP A1 is one of the most abundant and ubiquitously expressed members
(reviewed in [94]). Its role is not limited to splicing regulation, but includes func-
tions in transcription [95–97], mRNA stability [98, 99], mRNA export [100],
translation [101, 102], and telomere maintenance [103, 104]. Another example is
polypyrimidine-tract-binding protein (PTB) or hnRNP I (reviewed in [105]),
which is involved in splicing [106], mRNA stability [107], and polyadenylation
[108, 109]. It also stimulates translation initiation at picornavirus internal ribo-
some entry site (IRES) elements [110, 111]. HnRNP L contains four RRMs that
specifically recognize CA-repeat and CA-rich RNA elements [112]. It partici-
pates in intronless mRNA export [113, 114], translational regulation [98], mRNA
stability [112, 115], poly(A) site selection, and alternative splicing [112]. HnRNP
L competes with microRNAs for binding to a CA-rich RNA element within the
vegfa (vascular endothelial growth factor A) 3′ UTR [116]. Recently, activities of
hnRNP L were analyzed on a genome-wide level, and an in vivo enrichment of
CA motifs as hnRNP L binding sites was confirmed. A position-dependent splic-
ing regulation was demonstrated: while binding to intronic regions upstream of
alternative exons leads to repressed splicing, binding to the downstream intron
activates splicing [117].
Concerning their role as splicing regulators, many examples of hnRNPs and
hnRNP-like proteins show negative regulation, including Nova 1 [118], hnRNP
A1 [119], Fox2 [120], HuR [121], hnRNP H [122], hnRNP F [123], and PTB [124,
125]. A positive regulation has been shown for hnRNP A1 [126], hnRNP H [127],
hnRNP G [128], and PTB [129]. Similar to SR proteins, hnRNPs show a position-
dependent effect on splicing regulation [130].7.5.3 Mechanisms of Splicing Regulation
It is frequently difficult to make a clear distinction between “constitutive” and
“alternative” splicing. The decision depends on cis-acting elements like strong or
weak splice sites (a higher degree of similarity to the consensus sequence
increases splice site strength) and additional enhancer or silencer elements in the
vicinity of the splice sites. Furthermore, the abundance and concentration of
each splicing factor in a given cell type affects the splicing decision [131–133].
SR proteins are the main enhancers known to facilitate splice site recognition
and exon inclusion by binding to ESEs. In general, they help components of the
spliceosome to bind the pre-mRNA. This includes the recruitment of the U1