Peeking into the quality control art of cellular chefs
If you’ve ever enjoyed a meal, thank the chef—and if you’ve ever enjoyed life, credit spliceosomes, the cellular chefs of existence.
Genes in our DNA are like ingredient lists scattered across pages. Spliceosomes carefully remove non-coding introns and join coding exons to craft mature mRNAs, the blueprints for proteins.
Within every human cell, around 100000 spliceosomes are hard at work. But with this scale, mistakes can happen. Spliceosomes rely on quality control mechanisms to catch and correct errors. Faulty quality control has been linked to dire consequences, such as neurodevelopmental disorders, cancer, and other diseases.
“While it was known that spliceosomes have robust quality control mechanisms, very little was understood about how this process works,” says Associate Professor Tamás Fischer from The Australian National University (ANU), an expert in RNA surveillance.
To uncover the details, Fischer’s team specialised in RNA technologies collaborated with structural biologists at the Heidelberg University Biochemistry Center who captured high-resolution structures of a defective spliceosome on its way to disassembly.
Their findings, published in Nature Structural & Molecular Biology, provide fresh insight into how cells identify and discard faulty spliceosomes.
Freezing the frame
Spliceosomes are intricate cellular machines made of over 100 different proteins and five small nuclear RNAs. They assemble and disassemble step by step as they process pre-mRNA, making it technically challenging to capture transient intermediate states.
To tackle this, the researchers added small affinity tags to two proteins involved in the discard pathway and used them to selectively “fish out” defective spliceosomes from fission yeast (Schizosaccharomyces pombe) cells.
The researchers then froze these complexes using cryogenic electron microscopy (cryo-EM) to capture their structures in vivid detail.
“Our purification strategy, coupled with the largely rigid nature of the spliceosome core, facilitated the capture of high-resolution information for the core,” says co-corresponding author Professor Irmgard Sinning from Heidelberg University Biochemistry Center.
Analysing components flexibly attached to the periphery, however, posed a tougher challenge.
“To address this, we employed extensive particle sorting, rigorously tested various cryo-EM data analysis software suites, and complemented the structural data with crosslinking mass spectrometry in collaboration with Max Planck Institute for Multidisciplinary Sciences,” lead author Dr Komal Soni explains.
These strategies yielded two high-resolution reconstructions of defective spliceosomes at average resolutions of 3.2 and 3.1 angstroms (Å, 10-10 m)—close to atomic detail.
“These structures are the first that show a quality control arrested spliceosome in molecular details,” notes Fischer, who used RNA sequencing to unravel specific features of aberrant pre-mRNAs that led spliceosome to discard.
From yeast to human
These captured structures provide snapshots of a malfunctioning spliceosome stuck in the discard pathway.
Analysis revealed what goes wrong and how the spliceosome recognises errors, halts splicing, and moves towards disassembly.
From the structures, the study also identified two molecular models for this discard pathway, shedding light on the molecular choreography of this process.
The researchers believe that the recognition and discard mechanisms for defective spliceosomes in yeast are highly conserved.
“Each of the proteins involved in the process is conserved from fission yeast to humans,” says Fischer, highlighting the broader relevance of their findings.
The research is part of a long-standing collaboration between ANU and Heidelberg University to study the intricacies of RNA quality control.
“We want to understand how this basic mechanism works,” Fischer says, “Our next goal is to map the same process in human cells.”
By understanding these molecular ‘executive chefs’—as Fischer put it—scientists are edging closer to decoding life’s most intricate recipes, one splice at a time.