Cryo-EM structure of the CBC-ALYREF complex

Reviewer #1 (Public Review):

Summary:
The authors use a combination of biochemistry and cryo-EM studies to explore a complex between the cap-binding complex and an RNA binding protein, ALYREF, that coordinates mRNA processing and export.

Strengths:
The biochemistry and structural biology are supported by mutagenesis which tests the model in vitro. The structure provides new insight into how key events in RNA processing and export are likely to be coordinated.

Weaknesses:
The authors provide biochemical studies to confirm the interactions that they identify; however, they do not perform any studies to test these models in cells or explore the consequences of mRNA export from the nucleus. In fact, several of the amino acids that they identified in ALYREF that are critical for the interaction, as determined by their own biochemical studies, are conserved in budding yeast Yra1 (residues E124/E128 are E/Q in budding yeast and residues Y135/V138/P139 are F/S/P), where the impact on poly(A) RNA export from the nucleus could be readily evaluated. The authors could at least mention this point as part of the implications and the need for future studies. No one seems to have yet targeted any of these conserved residues, so this would be a logical extension of the current work.

Specific suggestions:
The authors could put their work in context by speculating how some of the amino acids that they identify as being critical for the interactions they identify could contribute to cancer. For example, they mention mutations of interacting residues in NCBP2 are associated with human cancers, pointing out that NCBP2 R105C amino acid substitution has been reported in colorectal cancer and the NCBP2 I110M mutation has been found in head and neck cancer. Do the authors speculate that these changes would decrease the interaction between NCBP2 and ALYREF and, if so, how would this contribute to cancer? They also mention that a K330N mutation in NCBP1 in human uterine corpus endometrial carcinoma, where Y135 on the α2 helix of mALYREF2 makes a hydrogen bond with K330 of NCBP1. How do they speculate loss of this interaction would contribute to cancer?

Reviewer #2 (Public Review):

Summary:
In this manuscript, Bradley and his colleagues represented the cryo-EM structure of the nuclear cap-binding complex (CBC) in complex with an mRNA export factor, ALYREF, providing a structural basis for understanding CBC regulating gene expression.

Strengths:
The authors successfully modeled the N-terminal region and the RRM domain of ALYREF (residues 1-183) within the CBC-ALYREF structure, which revealed that both the NCBP1 and NCBP2 subunits of the CBC interact with the RBM domain of ALYREF. Further mutagenesis and pull-down studies provided additional evidence to the observed CBC-ALYREF interface. Additionally, the authors engaged in a comprehensive discussion regarding other cellular complexes containing CBC and/or ALYREF components. They proposed potential models that elucidated coordinated events during mRNA maturation. This study provided good evidence to show how CBC effectively recruits mRNA export factor machinery, enhancing our understanding of CBC regulating gene expression during mRNA transcription, splicing, and export.

Weaknesses:
No in vivo or in vitro functional data to validate and support the structural observations and the proposed models in this study. Cryo-EM data processing and structural representation need to be strengthened.

Reviewer #3 (Public Review):

Summary:
The authors carried out structural and biochemical studies to investigate the multiple functions of CBC and ALYREF in RNA metabolism.

Strengths:
For the structural study part, the authors successfully revealed how NCBP1 and NCBP2 subunits interact with mALYREF (residues 1-155). Their binding interface was then confirmed by biochemical assays (mutagenesis and pull-down assays) presented in this study.

Weaknesses:
The authors did not provide functional data to support their proposed models. The authors should include more details regarding the workflow of their cryo-EM data processing in the figure.