C9ORF78

C9ORF78 is an intrinsically unstructured nuclear protein that functions as a spliceosome-associated regulator of alternative splicing and participates in mitotic chromosome segregation. Cryo-EM structures show that C9ORF78 wraps around the C-terminal helicase cassette of the spliceosome remodeling factor BRR2 in a manner mutually exclusive with FBP21, and can simultaneously contact PRPF22, establishing it as a late-stage splicing regulator at a multi-factor trafficking site; its depletion alters NAGNAG 3′-splice site usage and causes BRR2-dependent exon skipping (PMID:35241646). This spliceosomal role is conserved: the fission yeast orthologue Tls1 associates with Brr2 and regulates splicing of shelterin component mRNAs, with consequent effects on telomere integrity (PMID:25245948). Beyond splicing, C9ORF78 partially localizes to centromeres/kinetochores during mitosis and is required for proper chromosome segregation (PMID:35167828), and it interacts with FAM50A via its S121 residue to enhance ASNS transcription and asparagine biosynthesis (PMID:40531994).

1. 2014 Medium

   Identification of the fission yeast orthologue Tls1 as a Brr2-associated splicing factor established that C9ORF78 family proteins regulate pre-mRNA splicing with downstream consequences for chromatin and telomere homeostasis.

   Evidence Affinity purification/mass spectrometry of Tls1 interactors, RNA-seq of splicing changes, and genetic rescue in S. pombe, with supporting human spliceosome association data

   PMID:25245948

   Open questions at the time

   • Mechanism of C9ORF78–BRR2 interaction in human cells was unknown
   • No structural information on how C9ORF78 contacts BRR2
   • Whether C9ORF78 regulates the same splicing targets as Tls1 in human cells was untested
2. 2022 High

   Cryo-EM structures and functional studies resolved how C9ORF78 binds BRR2 at a multi-factor trafficking site in a mutually exclusive manner with FBP21, directly linking this interaction to regulation of alternative splicing including NAGNAG 3′-splice site choice and exon skipping.

   Evidence Cryo-EM structure determination, in vitro binding assays, RNA UV-crosslinking, affinity purification/mass spectrometry, and siRNA knockdown with splicing readout in human cells

   PMID:35241646

   Open questions at the time

   • How C9ORF78-to-FBP21 exchange is temporally regulated during the splicing cycle is unknown
   • Whether C9ORF78 catalytically modulates BRR2 helicase activity or only serves as a scaffold is unresolved
   • Full repertoire of C9ORF78-dependent splicing events genome-wide not defined
3. 2022 Medium

   Discovery of C9ORF78 at centromeres/kinetochores during mitosis revealed a second, non-spliceosomal function in chromosome segregation, with expression under cell-cycle and oncogenic transcription factor control.

   Evidence GFP tagging, immunofluorescence co-localization with ACA/Mad1/Ndc80, siRNA knockdown with mitotic phenotype scoring, E2F1/N-Myc overexpression

   PMID:35167828

   Open questions at the time

   • Molecular mechanism by which C9ORF78 promotes faithful chromosome segregation is unknown
   • Whether the kinetochore function is independent of splicing regulation or secondary to it has not been dissected
   • Direct kinetochore-binding partners of C9ORF78 have not been identified
4. 2025 Medium

   Identification of the FAM50A–C9ORF78 interaction at residue S121 revealed an unexpected role in transcriptional activation of ASNS and asparagine biosynthesis, linking C9ORF78 to metabolic reprogramming.

   Evidence Co-immunoprecipitation, S121 site-directed mutagenesis, ASNS transcription and asparagine synthesis assays, in vivo brain metastasis model

   PMID:40531994

   Open questions at the time

   • Whether S121 is a phospho-regulatory site that controls the FAM50A interaction is unknown
   • How a spliceosome-associated protein activates transcription of a specific gene (ASNS) mechanistically is unresolved
   • Whether this function operates in normal physiology or only in cancer contexts is untested

• It remains unknown how C9ORF78's spliceosomal, kinetochore, and transcriptional functions are coordinated across the cell cycle, and whether they share a common structural or regulatory basis.
• No separation-of-function mutants distinguishing splicing, kinetochore, and transcription roles
• No in vivo animal model for C9ORF78 loss-of-function phenotype
• Whether BRR2 helicase activity is modulated by C9ORF78 binding has not been measured biochemically