Structural Insights into Spliceosome Fidelity: DHX35–GPATCH1-Mediated Rejection of Aberrant Splicing Substrates

Academic Background Introduction

The spliceosome is a highly dynamic macromolecular complex responsible for the precise excision of introns from pre-mRNA. Although recent advances in cryo-electron microscopy (cryo-EM) have provided a comprehensive structural understanding of the stepwise assembly, catalytic splicing, and final disassembly of the spliceosome, the molecular mechanisms by which the spliceosome recognizes and rejects suboptimal splicing substrates remain unclear. This issue is crucial for understanding splicing fidelity, as splicing errors can lead to abnormal gene expression and various diseases.

This study aims to reveal how the spliceosome recognizes and rejects aberrant splicing substrates, particularly those containing non-canonical 5’ splice sites (5’SS), through specific RNA helicases and G-patch proteins. By investigating spliceosomal quality control complexes from the thermophilic eukaryote Chaetomium thermophilum, the authors provide high-resolution cryo-EM structures, elucidating the critical role of the DHX35–GPATCH1 complex in spliceosome fidelity.

Source of the Paper

The paper was co-authored by Yi Li, Paulina Fischer, Mengjiao Wang, and others from multiple institutions including Fudan University, Heidelberg University Biochemistry Center (BZH), and Max-Planck-Institute for Multidisciplinary Sciences. It was published in 2025 in the journal Cell Research, titled “Structural insights into spliceosome fidelity: DHX35–GPATCH1-mediated rejection of aberrant splicing substrates”.

Research Process and Results

1. Research Process

a) Isolation and Purification of the Spliceosome

The research team first isolated spliceosome complexes from Chaetomium thermophilum. Using the conserved disassembly factor DHX15 as bait, the researchers successfully purified multiple spliceosome core proteins and known disassembly factors through tandem-affinity purification. Mass spectrometry further confirmed the co-precipitation of spliceosomal proteins with DHX15, including U2 snRNP and U5 snRNP.

b) Cryo-EM Data Collection and Processing

The researchers collected cryo-EM data and, through automated particle picking and 3D classification, resolved three major spliceosome complexes: the ILS complex (intron-lariat spliceosome) and two quality control complexes, B*Q1 and B*Q2. These complexes represent states where the spliceosome is intercepted after catalytic activation but before the first splicing reaction.

c) Structural Modeling and Analysis

By combining AlphaFold-predicted protein structures with cryo-EM density maps, the researchers constructed molecular models of the spliceosome. In particular, they detailed the interactions between the DHX35–GPATCH1 complex and the spliceosome, revealing how GPATCH1 recognizes aberrant 5’SS through its G-patch domain and anchors DHX35 to the spliceosome.

2. Main Results

a) Structure of the DHX35–GPATCH1 Complex

The results show that GPATCH1 interacts with multiple domains of the spliceosomal core protein PRP8 (including the EN, RH, and α-finger domains) through its N-terminal region, thereby anchoring DHX35 to the spliceosome. The G-patch domain of GPATCH1 binds to the WH and RecA2 domains of DHX35, ensuring its RNA helicase activity. Additionally, GPATCH1 guides DHX35 to its substrate by interacting with U2 snRNA.

b) Helicase Mechanism of DHX35

In the B*Q complexes, DHX35 binds to the single-stranded region of U2 snRNA and dissociates the U2/branch site (BS) helix. This process prevents further catalytic reactions of the spliceosome, ensuring the rejection of aberrant splicing substrates. The helicase activity of DHX35 depends on the activation by GPATCH1, which enhances DHX35’s ATPase activity through its G-patch domain.

c) Disassembly Role of DHX15

In the B*Q2 complex, DHX15 is recruited to a binding site similar to that in the ILS complex and is positioned close to its substrate, the 3’ end of U6 snRNA. This suggests that DHX15 plays a key role in the disassembly of the spliceosome by unwinding U6 snRNA to promote its final disassembly.

3. Research Conclusions

This study, through high-resolution cryo-EM structures, reveals the critical role of the DHX35–GPATCH1 complex in spliceosome fidelity. GPATCH1 recognizes aberrant 5’SS and anchors DHX35 to the spliceosome, ensuring the rejection of aberrant splicing substrates. Meanwhile, DHX15 plays an important role in spliceosome disassembly by unwinding U6 snRNA to promote its final disassembly. These findings provide new structural insights into spliceosome quality control mechanisms and offer important clues for understanding diseases related to splicing errors.

4. Research Highlights

• High-Resolution Structural Analysis: For the first time, high-resolution structures of the DHX35–GPATCH1 complex with the spliceosome were resolved using cryo-EM.
• Spliceosome Fidelity Mechanism: Revealed how GPATCH1 recognizes aberrant 5’SS and anchors DHX35 to the spliceosome, ensuring the rejection of aberrant splicing substrates.
• Dual Helicase Synergy: Proposed a synergistic mechanism of DHX35 and DHX15 in spliceosome quality control, providing new insights into the spliceosome disassembly process.

5. Other Valuable Information

This study also validated the function of DHX35 and GPATCH1 in spliceosome quality control through RNA sequencing. The researchers found that knockdown of DHX35 or GPATCH1 increased splicing events of pre-mRNAs containing suboptimal 5’SS, further supporting their critical role in splicing fidelity.

Summary

Through high-resolution cryo-EM structures and functional experiments, this study reveals the critical role of the DHX35–GPATCH1 complex in spliceosome fidelity. These findings not only provide new structural insights into spliceosome quality control mechanisms but also offer important clues for research on diseases related to splicing errors.