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\b[Shared preference for pyrimidine-rich <RNA> does not prescribe one splicing outcome: position, multivalent binding and interactions with the <spliceosome> determine the regulated step.] <Sex-lethal> acts in the Drosophila sex-determination programme, whereas <PTBP1> regulates many mammalian precursor RNAs in a cell-type-dependent manner. Their effects must be interpreted against competing <splice sites> and the <proteins> normally recognizing them.
During early <spliceosome> assembly, <U1 snRNP> recognizes a 5′ <splice site>, while factors including <U2AF> help recognize a <polypyrimidine tract> and 3′ splice-site region and promote assembly around the branch point. Alternative sites compete within this context. Binding of another <RNA-binding protein> can occlude an essential interaction, organize a repressive complex or favour use of a competing site; recognition need not be determined solely by an isolated site's consensus strength.
A particularly clear <SXL> mechanism operates on precursor <RNA> encoding <Transformer protein>. In the absence of <SXL>, <U2AF> recognizes a strong default 3′ site, producing an unproductive transcript. In female <biological cells>, <SXL> binds the pyrimidine-rich region at that site and competes with <U2AF>. The weaker alternative female site can then be used, producing functional <Transformer protein>. Reconstituted splicing experiments showed the antagonism, and adding a <U2AF> effector domain to <SXL> converted its activity from repression toward activation.<The protein Sex-lethal antagonizes the splicing factor U2AF to regulate alternative splicing of transformer pre-mRNA by Juan Valcárcel and others>[In vitro splice-site switch and effector-domain fusion]{ref} The domain-fusion result is important: related <RNA> recognition can direct different outcomes because the <proteins> recruit different downstream activities. <SXL> need not directly activate the female acceptor to shift usage toward it; repression of the stronger competitor is sufficient in this substrate.
<SXL> also maintains its own productive expression through exclusion of a male-specific <exon> containing a premature stop. This provides positive feedback in female <biological cells>. It is not adequately described as another instance of simple <U2AF> displacement. Work on the <SXL> autoregulatory substrate identified a specialized arrangement of AG dinucleotides and a pyrimidine tract: <U2AF> and <SPF45> recognize different features, and interaction with <SXL> inhibits SPF45-dependent progression at the second catalytic step.<Splicing regulation at the second catalytic step by Sex-lethal involves 3′ splice site recognition by SPF45 by María José Lallena and others>[Autoregulatory exon 3 and second-step inhibition]{ref} <SXL> can consequently alter site selection after early assembly has occurred. Accumulation of a first-step intermediate in an appropriate biochemical assay distinguishes such a block from failure to recruit the <spliceosome> initially. The same regulator can use different mechanisms on different RNAs.
<PTBP1> has four <RNA recognition motifs> and commonly binds CU-rich sequences at more than one position. Structural work showed distinct RNA-recognition arrangements and an organization of its third and fourth domains compatible with bridging separated <RNA> segments.<Structure of PTB bound to RNA: specific binding and implications for splicing regulation by Florian C. Oberstrass and others>[RNA-bound domain structures and implications for recognition]{ref} <Förster resonance energy transfer>, <NMR> and splicing assays subsequently supported <RNA looping> by these domains: contacts on either side of a segment can organize an <RNA> geometry that disfavors <exon> use.<RNA looping by PTB: evidence using FRET and NMR spectroscopy for a role in splicing repression by Rajan Lamichhane and others>[Looping measurements and functional tests]{ref} These observations support a physical repression mechanism, but a short model <RNA> loop does not prove that every endogenous PTBP1-regulated <exon> is looped out.
Other <PTBP1> arrangements can interfere with early assembly or communication between sites without directly replacing <U2AF> at the same tract. Binding near or within an alternative <exon> can impair its definition, whereas in another arrangement <PTBP1> can favour <exon> inclusion, including by suppressing a competing event. Genome-wide perturbation and positional analyses revealed both activation and repression associated with different binding contexts.<Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB by Miriam Llorian and others>[Global splicing responses and position-dependent activity]{ref} Thus neither “all pyrimidine binders are repressors” nor “<PTBP1> always causes skipping” is a valid general rule. <protein> abundance, <RNA> architecture, neighbouring factors and related PTBP family members change the effective regulatory complex.
To discriminate mechanisms, map direct contacts by <crosslinking and immunoprecipitation>, measure exon-junction usage after acute depletion, and rescue with wild-type or selected RNA-binding/interface mutants. Use a <minigene splicing reporter> to mutate individual tracts, change their spacing or move them relative to a regulated <exon>, then confirm important effects at the endogenous locus. Assays of <U2AF> or <U1 snRNP> association and native spliceosomal assembly can reveal an early block; time-resolved splicing-intermediate analysis can reveal a late one. Control reporter <transcription> and precursor abundance, and account for <nonsense-mediated decay>, because an unstable mature isoform can conceal the splice choice that generated it. Mutants must retain expression and the intended remaining contacts. \b[<SXL> supplies examples of both competition and late catalytic control; <PTBP1> illustrates multivalent, context-dependent organization of splice-site recognition. <RNA> affinity is only one part of specificity.]
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