Biology subjects

BUTCHER, S.

Publications and source records attributed to BUTCHER, S..

3 recordsLinked to original sources

bioRxiv2025

TDP-43 controls RNA structure through high affinity lattice interactions

TDP-43 is an RNA binding protein implicated in neurodegenerative disease. TDP-43 binds to GU dinucleotide repeats, which are highly abundant sequences in human RNA. Here we show TDP- 43 has one of the highest affinities and specificities measured for an RNA binding protein. Binding prevents formation of the pUG fold, an intramolecular quadruplex, and conversely pUG fold formation prevents TDP-43 binding. A rapid on-rate allows TDP-43 to capture single stranded RNA prior to folding. The protein recognizes the RNA as a 1D lattice, in which overlapping binding sites produce efficient initial binding events that interfere with subsequent interactions. This effect is partially overcome by RNA facilitated protein-protein interactions, which serve to increase the on-rate of a second TDP-43 molecule. In conjunction with all atom models, these data reveal how TDP-43 recognizes RNA repeat sequences and identify an interplay between RNA folding and protein recognition that may be relevant to human disease.

biochemistry↗

bioRxiv2025

RNA Modifications and Prp24 Coordinate Lsm2-8 Binding Dynamics during S. cerevisiae U6 snRNP Assembly

In eukaryotes, the process of intron removal from nuclear pre-mRNA is performed by the spliceosome, a dynamic molecular machine composed of small nuclear ribonucleoproteins (snRNPs; U1, U2, U4, U5, and U6) and dozens of other protein splicing factors. The U6 snRNP contains the U6 snRNA and the proteins Prp24 and Lsm2-8 heteroheptamer. A key feature of the snRNP is a modified U6 snRNA 3 end, which in S. cerevisiae (yeast) contains a 3 phosphate. U6 plays an essential role in splicing, and the U6 snRNP must be completely disassembled for splicing to occur. Once splicing is finished, the snRNP must then be reassembled to participate in a subsequent splicing reaction. While splicing efficiency depends on rapid U6 snRNP assembly, this process has not yet been kinetically characterized. Here, we use colocalization single molecule spectroscopy (CoSMoS) to dissect the kinetic pathways of yeast U6 snRNA association with the Lsm2-8 complex and their dependence on the Prp24 protein and post-transcriptional snRNA modification. In the absence of 3 end processing, Lsm2-8 association with the RNA is highly dependent on Prp24. However, processed RNAs can rapidly recruit Lsm2-8 in Prp24s absence. Post-transcriptional processing facilitates Lsm2-8 association while the presence of Prp24 promotes both recruitment and retention of the complex. This suggests that efficient U6 snRNP assembly could depend on kinetic selection of Lsm2-8 binding to 3-end modified or Prp24 bound U6 snRNAs in order to discriminate against association with other RNAs.

biochemistry↗

bioRxiv2021

An Atypical RNA Quadruplex Marks RNAs as Vectors for Gene Silencing

The addition of poly(UG) ("pUG") repeats to 3' termini of mRNAs drives gene silencing and trans-generational epigenetic inheritance in the metazoan C. elegans.1 pUG tails promote silencing by recruiting an RNA-dependent RNA Polymerase (RdRP) that synthesizes small interfering (si)RNAs.1 Here we show that active pUG tails require a minimum of 11.5 repeats and adopt a quadruplex (G4)2 structure we term the pUG fold. The pUG fold differs from known G4s in that it has a left-handed backbone similar to Z-RNA3,4, no consecutive guanosines in its sequence, and three G quartets and one U quartet stacked non-sequentially. Its biological importance is emphasized by our observations that porphyrin molecules bind to the pUG fold and inhibit both gene silencing and binding of RdRP. Moreover, specific N7-deaza RNA substitutions that do not adopt the pUG fold neither bind RdRP nor induce RNA silencing. These data define the pUG fold as a previously unrecognized RNA secondary structure motif that drives gene silencing. The pUG fold can also form internally within larger RNA molecules. Approximately 20,000 pUG-fold sequences are found in non-coding regions of human RNAs, suggesting the fold likely has biological roles beyond gene silencing.

molecular biology↗