Unraveling the mechanism of recognition of the 3’ splice site of the adenovirus major late promoter intron by the alternative splicing factor PUF60

Abstract

Pre-mRNA splicing is critical for achieving required amounts of a transcript at a given time and for regulating production of encoded protein. A given pre-mRNA may be spliced in many ways, or not at all, giving rise to multiple gene products. Numerous splicing factors are recruited to pre-mRNA splice sites to ensure proper splicing. One such factor, the 60 kDa poly(U)-binding splicing factor (PUF60), is recruited to sites that are not always spliced, but rather function as alternative splice sites. In this study, we characterized the interaction of PUF60 with a splice site from the adenovirus major late promoter (the AdML 3' splice site, AdML3’). We found that the PUF60–AdML3’ dissociation constants are in the micromolar range, with the binding affinity predominantly provided by PUF60’s two central RNA recognition motifs (RRMs). A 1.95 Å crystal structure of the two PUF60 RRMs in complex with AdML3’ revealed a dimeric organization placing two stretches of nucleic acid tracts in opposing directionalities, which can cause looping of nucleic acid and explain how PUF60 affects pre-mRNA geometry to effect splicing. Solution characterization of this complex by light-scattering and UV/Vis spectroscopy suggested a potential 2:1 (PUF602:AdML3’) stoichiometry, consistent with the crystal structure. This work defines the sequence specificity of the alternative splicing factor PUF60 at the pre-mRNA 3’ splice site. Our observations suggest that control of pre-mRNA directionality is important in the early stage of spliceosome assembly, and advance our understanding of the molecular mechanism by which alternative and constitutive splicing factors differentiate among 3’ splice sites.

Structural insights into a regulatory mechanism of FIR RRM1–FUSE interaction

“…We have provided a high-resolution model of the FIR RRM1-2-FUSE interaction and presented a comprehensive structural analysis of RRM-ssDNA/RNA interactions. We observed distinct binding modes of different RRM-ssDNA interactions and no binding-induced RRM1-2 protein-protein interaction as postulated previously [11,21]. Together with ITC binding data, our structural data demonstrated that FIR RRM1 has the potential to interact with diverse ssDNA sequences, consistent with previous data that reported on the low specificity of this protein [11].…”

“…Together with ITC binding data, our structural data demonstrated that FIR RRM1 has the potential to interact with diverse ssDNA sequences, consistent with previous data that reported on the low specificity of this protein [11]. Along with the previous structural studies [11,21], our crystal structures, which present different binding modes of the two bound dinucleotides, extend the repertoire of structural models for nucleotide interactions in FIR. The structural plasticity observed highlights the versatility of FIR RRMs in accommodating diverse oligonucleotide sequences, in agreement with published binding studies [11,21].…”

“…Along with the previous structural studies [11,21], our crystal structures, which present different binding modes of the two bound dinucleotides, extend the repertoire of structural models for nucleotide interactions in FIR. The structural plasticity observed highlights the versatility of FIR RRMs in accommodating diverse oligonucleotide sequences, in agreement with published binding studies [11,21]. Overall, our observations demonstrate the dynamic nature of the RRM1-FUSE interface, which is consistent with an earlier study that suggested RRM as a versatile ssDNA/RNA-binding protein module contributing to a wide spectrum of nucleic acid sequence recognition [4].…”

Decreased spliceosome fidelity and egl-8 intron retention inhibit mTORC1 signaling to promote longevity

“…4a,b ). This residue mediates interdomain RRM1–RRM2 contacts in the PUF60 crystal structure 16 and may affect salt bridge formation. Rbm-39 encodes an RNA-binding protein, the human ortholog of which, RBM39, functions as a splicing factor and is involved in early spliceosome assembly 17 .…”

Decreased spliceosome fidelity inhibits mTOR signalling and promotes longevity via an intron retention event

“…4a, b). This residue mediates interdomain RRM1-RRM2 contacts in the PUF60 crystal structure 14 , and may affect salt bridge formation. RBM-39 encodes an RNA binding protein, whose human ortholog, RBM39, functions as a splicing factor and is involved in early spliceosome assembly 15 .…”