Biology subjects

Spike, C. A.

Publications and source records attributed to Spike, C. A..

3 recordsLinked to original sources

bioRxiv2025

The SPN-4 Rbfox RNA-binding protein selects maternal mRNAs for CCR4-NOT-dependent clearance in early Caenorhabditis elegans embryos

The elimination of maternal mRNAs is an essential feature of the maternal-to-zygotic transition. We report an essential pathway that clears many maternal transcripts from early C. elegans embryos using the Rbfox-related SPN-4 RNA-binding protein as a specificity factor and the CCR4-NOT deadenylase complex as an effector. We biochemically identified SPN-4-associated mRNAs from late-stage oocytes and found that the set of SPN-4-associated transcripts is enriched for maternal mRNAs that undergo early decay. Single-molecule fluorescence in situ hybridization experiments established that many SPN-4-associated mRNAs fail to be eliminated in the absence of SPN-4. In the 3UTRs of two target mRNAs, we identified Rbfox motifs that are required for SPN-4-dependent clearance in vivo and bind SPN-4 in vitro. In a genetic screen to identify factors that work with SPN-4, we isolated mutant alleles of CCR4-NOT components. Auxin-induced degradation of the LET-711/NOT1 scaffold and the CCF-1 deadenylase disrupted clearance of two SPN-4-associated transcripts. Our results support a model in which SPN-4 initiates expression in late-stage oocytes, associates with maternal mRNA targets through RNA sequences in their 3UTRs and promotes CCR4-NOT-mediated decay during early embryogenesis.

developmental biology↗

bioRxiv2021

Ubiquitin ligases and a processive proteasome facilitate protein clearance during the oocyte-to-embryo transition in Caenorhabditis elegans

The ubiquitin-mediated degradation of oocyte translational regulatory proteins is a conserved feature of the oocyte-to-embryo transition (OET). In the nematode Caenorhabditis elegans, multiple translational regulatory proteins, including the TRIM-NHL RNA-binding protein LIN-41/Trim71 and the Pumilio-family RNA-binding proteins PUF-3 and PUF-11, are degraded during the OET. Degradation of each protein requires activation of the M-phase cyclin-dependent kinase CDK-1, is largely complete by the end of the first meiotic division and does not require the anaphase promoting complex (APC). However, only LIN-41 degradation requires the F-box protein SEL-10/FBW7/Cdc4p, the substrate recognition subunit of an SCF-type E3 ubiquitin ligase. This finding suggests that PUF-3 and PUF-11, which localize to LIN-41-containing ribonucleoprotein particles (RNPs), are independently degraded through the action of other factors and that the oocyte RNPs are disassembled in a concerted fashion during the OET. We develop and test the hypothesis that PUF-3 and PUF-11 are targeted for degradation by the proteasome-associated HECT-type ubiquitin ligase ETC-1/UBE3C/Hu15, which is broadly expressed in C. elegans. We find that several GFP-tagged fusion proteins that are degraded during the OET, including fusions with PUF-3, PUF-11, LIN-41, IFY-1/Securin and CYB-1/Cyclin B, are incompletely degraded when ETC-1 function is compromised. However, it is the fused GFP moiety that appears to be the critical determinant of this proteolysis defect. These findings are consistent with a conserved role for ETC-1 in promoting proteasome processivity and suggest that proteasomal processivity is an important element of the OET during which many key oocyte regulatory proteins are rapidly targeted for degradation. Article SummaryThe ubiquitin-mediated degradation of translational regulatory RNA-binding proteins is a conserved feature of the oocyte-to-embryo transition (OET). C. elegans LIN-41 is a master regulator of oogenesis and is found in a large translational regulatory ribonucleoprotein (RNP) complex with more than 1000 maternal transcripts and the Pumilio-family RNA-binding proteins PUF-3 and PUF-11. We show that the concerted action of ubiquitin-mediated protein degradation and proteasome processivity rapidly disassemble LIN-41-containing RNPs during the OET thereby relieving repression of many maternal transcripts.

developmental biology↗

bioRxiv2019

Insights into the involvement of spliceosomal mutations in myelodysplastic disorders from an analysis of SACY-1/DDX41 in Caenorhabditis elegans

Mutations affecting spliceosomal proteins are frequently found in hematological malignancies, including myelodysplastic syndromes and acute myeloid leukemia. DDX41/Abstrakt is a metazoan-specific spliceosomal DEAD-box RNA helicase found to be recurrently mutated in inherited myelodysplastic syndromes and in relapsing cases of acute myeloid leukemia. The genetic properties and genomic impacts of disease-causing missense mutations in DDX41 and other spliceosomal proteins have been uncertain. Here we conduct a comprehensive molecular genetic analysis of the C. elegans DDX41 ortholog, SACY-1. Our results reveal general essential functions for SACY-1 in both the germline and the soma, as well as specific functions affecting germline sex determination and cell cycle control. Certain sacy-1/DDX41 mutations, including the R525H human oncogenic variant, confer antimorphic activity, suggesting that they compromise the function of the spliceosome. Consistent with these findings, sacy-1 exhibits synthetic lethal interactions with several spliceosomal components, and biochemical analyses suggest that SACY-1 is a component of the C. elegans spliceosome. We used the auxin-inducible degradation system to analyze the impact of SACY-1 on the transcriptome using RNA sequencing. SACY-1 depletion impacts the transcriptome through splicing-independent and splicing-dependent mechanisms. The observed transcriptome changes suggest that disruption of spliceosomal function induces a stress response. Altered 3 splice site usage represents the predominant splicing defect observed upon SACY-1 depletion, consistent with a role for SACY-1 as a second-step splicing factor. Missplicing events appear more prevalent in the soma than the germline, suggesting that surveillance mechanisms protect the germline from aberrant splicing. Author SummaryMutations affecting spliceosomal proteins are frequently found in hematological malignancies. DDX41/Abstrakt is a metazoan-specific spliceosomal DEAD-box RNA helicase recurrently mutated in inherited and relapsing myelodysplastic syndromes and acute myeloid leukemia. The genetic properties and genomic impacts of disease-causing mutations in spliceosomal proteins have been uncertain. Here we conduct a comprehensive molecular genetic analysis of the C. elegans DDX41 ortholog, SACY-1. Our results reveal that multiple sacy-1/DDX41 missense mutations, including the R525H human oncogenic variant, exhibit antimorphic activity that likely compromises the function of the spliceosome. The genomic consequences of SACY-1 depletion include splicing-splicing-independent and splicing-dependent alterations in the transcriptome.

genetics↗