Past exam of the biochemistry course of the University of Cambridge / 2024 / Paper 2 / Section II / 4 / Solution

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.^[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.^[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.^[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.^[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.^[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. 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.