The article titled “Hominini-specific regulation of the cell cycle by stop codon readthrough of FEM1B” explores the regulation of FEM1B expression by stop codon readthrough (SCR), which leads to the generation of a C-terminally extended isoform that has been identified as highly unstable. The research highlights a crucial 81-nucleotide cis-signal in the proximal 3′ untranslated region (UTR) of FEM1B, responsible for SCR and encoding the amino acid sequence accountable for the degradation of the SCR product.
The article sheds light on the intricate regulation of the cell cycle by focusing on the stop codon readthrough mechanism involving FEM1B. This protein is a substrate-recognition component of the CRL2 E3 ubiquitin-protein ligase, which targets specific proteins for ubiquitylation and subsequent degradation. The authors present compelling evidence demonstrating how the SCR of FEM1B impacts the cell cycle regulation, emphasizing the Hominini-specific aspects of this mechanism.
Results
The study’s results elucidate the pivotal role of stop codon readthrough in shaping the regulation of the cell cycle, particularly in the context of Hominini-specific biology. The identification of the 81-nucleotide cis-signal in the 3′ UTR of FEM1B as the critical element for SCR underscores the specificity and precision of this regulatory process. Furthermore, the identified mechanism enhances our understanding of how the cell cycle is intricately controlled and provides insights into the evolutionary adaptations in Hominini.
Discussion
The implications of the findings presented in this article are profound, as they provide a platform for further investigations into the regulatory mechanisms governing the cell cycle in Hominini. The discussion encompasses the potential impact of SCR on cellular processes, the evolutionary significance of Hominini-specific regulation, and the broader implications for understanding cellular dynamics and adaptations in primates.
Thus, the article “Hominini-specific regulation of the cell cycle by stop codon readthrough of FEM1B” presents a significant contribution to our understanding of the regulatory mechanisms governing the cell cycle, specifically in the context of Hominini biology. The authors provide compelling evidence supporting the impact of SCR on FEM1B expression and its subsequent influence on cellular processes. This research has the prospect of catalyzing further inquiries into the nuanced regulation of the cell cycle, and its evolutionary implications in Hominini.
Here is the article Summary:
Title: Regulation of FEM1B Expression:
– FEM1B expression is regulated by stop codon readthrough (SCR).
– Translating ribosomes readthrough the stop codon of FEM1B to generate an unstable C-terminally extended isoform.
Title: Function of SCR in FEM1B Regulation:
– 81 nucleotides in the proximal 3′UTR of FEM1B are essential for SCR.
– These nucleotides encode the amino acid sequence responsible for the instability of the SCR product.
Title: Impact on Cell Cycle:
– CRISPR-edited cells lacking the SCR region of FEM1B show increased FEM1B expression.
– Increased FEM1B expression leads to reduced levels of SLBP and replication-dependent histones, causing cell cycle delay.
Title: Evolutionary Analysis:
– The SCR process regulating the cell cycle by FEM1B is specific to the genus Pan and Homo (Hominini).
– This phenomenon represents a relatively recent evolutionary adaptation.
CRL2FEM1B-Mediated Protein Degradation:
– The FEM1B-containing version of CRL2 targets proteins like CDK5R1, FNIP1, GLI1, ANKRD37, SMCR8, and SLBP for ubiquitylation and degradation.
– This regulatory mechanism influences cellular functions including redox balance, oncogenicity, and cell cycle progression.
Role of FEM1B in Various Biological Processes:
– FEM1B, along with FEM1A and FEM1C, acts as a substrate-recognition component of CRL2.
– Polymorphisms in FEM1B are linked to conditions like polycystic ovary syndrome, and its absence in mice affects glucose tolerance.
Functional Insights and Regulation of CRL2:
– CRL2 has a crucial role in cellular functions and is regulated by the COP9 signalosome complex.
– CRL2FEM1B regulation involves the newly discovered mechanism involving stop codon readthrough during FEM1B translation.
Significance of Stop Codon Readthrough (SCR):
– SCR is a translation process that can generate protein isoforms with extended C-termini.
– This process is observed at varying frequencies and can impact protein function, localization, and stability.
Functional Implications of SCR in FEM1B:
– SCR in FEM1B mRNA generates an isoform susceptible to rapid degradation in humans and chimpanzees.
– Cells lacking this SCR exhibit reduced proliferation, clonogenicity, and tumorigenicity due to enhanced FEM1B expression.
Importance of Proximal 3′UTR in FEM1B mRNA:
– Deletion experiments reveal that the proximal 3′UTR of FEM1B mRNA is critical for normal cell cycle progression.
– Conserved across mammals, this region influences proliferation, clonogenicity, and cell cycle dynamics.
Experimental Evidence and Cellular Impact:
– Experiments in HeLa and MDA-MB-231 cells support the role of the proximal 3′UTR in cell cycle regulation.
– Alterations in the proximal 3′UTR result in reduced proliferation, clonogenicity, and S-phase delays in cancer cell lines.
Functional Analysis of FEM1B mRNA:
– Characterizing the functional importance of FEM1B mRNA reveals insights into cell cycle control mechanisms.
– These findings emphasize the significance of specific mRNA regions in governing cellular processes.
3′UTR Deletion in HeLa Cells:
– Deletion of 3′UTR in HeLa cells resulted in a significant increase in the expression of FEM1B protein but no change in mRNA levels, indicating post-transcriptional regulation.
– Enhanced FEM1B expression in Δ3′UTR cells caused reduced proliferation, clonogenicity, and delay in the S-phase of the cell cycle.
FEM1B-Mediated Regulation:
– FEM1B-mediated degradation of SLBP led to reduced expression of replication-dependent histone proteins H2B and H4, which are essential for normal cell cycle progression.
– Overexpression of SLBP in Δ3′UTR cells restored proliferation comparable to wild-type cells, indicating the involvement of SLBP in cell cycle regulation.
Increased FEM1B Expression Affects S-Phase Progression:
– FEM1B expression was found to be increased in UTR cells, leading to reduced levels of SLBP, H2B, and H4 proteins crucial for normal S-phase progression.
– Cellular levels of FEM1B were significantly altered in wild-type and delta 3’UTR HeLa cells as shown by Western blot analysis.
Regulation of FEM1B Expression by Its Proximal 3’UTR:
– The amino acid sequence encoded by the proximal 3’UTR of FEM1B mRNA is highly conserved across mammals.
– Observations suggest that the proximal 3’UTR might undergo translation by SCR.
Experimental Verification of Stop Codon Readthrough in FEM1B mRNA:
– Fluorescence-based SCR assay demonstrated significant fluorescence in HeLa cells, indicating SCR across the canonical stop codon of FEM1B.
– Luminescence-based SCR assay further supported the occurrence of SCR, as observed by quantification of firefly luciferase expression.
In vitro analysis of FEM1B SCR:
– In vitro transcription and translation experiments in rabbit reticulocyte lysate confirmed significant luminescence from FEM1B SCR construct.
– Mutating the canonical stop codon to UGA showed greater stop codon readthrough (SCR) compared to other stop codons.
Analysis of mass spectrometry data:
– Mass spectrometry data revealed the unique peptide GLLITDSR in primary human testicular peritubular cells, providing strong evidence for SCR of endogenous FEM1B.
– Ribosome profiling data supported the evidence for SCR, showing ribosome footprints beyond the canonical stop codon.
Evaluation of ISR of FEM1B and SCR:
– The ISR of FEM1B alone was found to drive stop codon readthrough in a heterologous system, demonstrating its role in initiating SCR.
– Human cell and in vitro studies confirmed the efficiency of SCR induced by the ISR of FEM1B.
Identification of FEM1Bx and its instability:
– The SCR of FEM1B generated a highly unstable isoform named FEM1Bx, with limited expression in comparison to FEM1B.
– Cell treatment experiments showed the involvement of the ubiquitin-dependent proteasome system in the degradation of FEM1Bx.
Detection of Synthetic Peptide:
– Anti-FEM1Bx antibody detected the synthetic peptide VTGYVKSFNVVLKSKGL, but not a control peptide.
– The antibody could not detect the peptide when it was pre-incubated with it.
Detection of Overexpressed Exogenous SCR product:
– The anti-FEM1Bx antibody was able to detect the overexpressed exogenous SCR product FEM1Bx in HEK293 cells.
– This demonstrates the specificity of the antibody.
Detection of Endogenous FEM1Bx:
– FEM1Bx was undetectable in many cell lines, including HeLa cells, due to the poor stability of this isoform.
– However, the anti-FEM1Bx antibody detected a band between 75 and 63 kDa in HepG2 cells, showing evidence for the endogenous FEM1Bx.
Mutation of the Canonical Stop Codon of FEM1B:
– Mutation of the canonical stop codon of FEM1B reduced its protein levels by 50% in mutant cells compared to their parental wild-type cells.
– FEM1B mRNA levels increased in the mutant cells, suggesting possible feedback regulation at the transcriptional level.
Effect of Reduced FEM1B Levels on Cell Proliferation:
– Reduced levels of the canonical isoform of FEM1B did not affect the cell cycle in mutant cells compared to Δ3′UTR cells with reduced cell proliferation.
Activity of FEM1B Isoforms:
– FEM1B activity was investigated in FEM1BX628Q−/− cells, showing reduced levels of FEM1B isoforms.
– An increase in the relative fluorescence of GFP tagged with CRL2FEM1B degron was observed in cells with low FEM1B levels.
Regulation of FEM1B Expression by SCR:
– Results indicate the regulation of FEM1B expression by substrate competition for the CRL2FEM1B E3 ubiquitin ligase complex.
Induction of Protein Degradation by FEM1B ISR Sequence:
– The 27 amino acid sequence encoded in the FEM1B ISR was sufficient to induce the degradation of a protein when transfected in HeLa cells.
GFP Expression Levels:
– GFP with C-terminal extension had lower expression compared to GFP without extension.
– Insertion of a stop codon restored stability to GFP.
Proteasomal Degradation:
– Proteasomal complex is involved in the degradation of GFP-ISR.
– MG132 could rescue the instability of GFP-ISR.
Evolution of FEM1Bx:
– FEM1Bx is longer in humans and chimpanzees due to SCR compared to other mammals.
– Regulation of FEM1B expression by SCR evolved recently.
Functional Significance of SCR:
– Δ3′UTR cells showed enhanced FEM1B expression.
– Xenograft tumours from Δ3′UTR cells were slower growing.
Regulation of Cancer Progression:
– FEM1B expression levels impact cancer progression.
– High FEM1B expression correlates with reduced survival in cancer patients.
Regulation of Cell Cycle by SCR:
– SCR of FEM1B mRNA controls FEM1B levels.
– SCR in humans and chimpanzees results in an unstable isoform (FEM1Bx).
Tumour growth monitoring:
– Tumour growth derived from HeLa cells (WT and Δ3′UTR) was monitored in athymic nude mice using digital Vernier callipers.
– The tumour progression was calculated by measuring the tumour volume and the weight of tumours after 30 days.
Survival analysis:
– Kaplan–Meier curves revealed the correlation between the probability of survival of cancer patients and the expression of FEM1B or SLBP.
– Higher expression of FEM1B showed a significant increase in the probability of survival in breast cancer patients.
Cell cycle regulation by SCR:
– Regulation of cell cycle by stop codon readthrough (SCR) of FEM1B mRNA was observed.
– SCR of FEM1B mRNA leads to an unstable isoform termed FEM1Bx, which affects the progression of S-phase of the cell cycle.
3′UTR’s role in mRNA expression:
– The 3′UTR of FEM1B plays a crucial role in regulating its expression.
– The proximal 3′UTR of FEM1B translates SCR, generating a longer unstable isoform prone to degradation.
Evolution of SCR in FEM1B:
– SCR-mediated regulation of FEM1B expression is present only in humans and chimpanzees.
– A single nucleotide insertion in the proximal 3′UTR of FEM1B in these primates partly explains their susceptibility to cancer.
Clinical significance of FEM1B and SLBP:
– FEM1B and SLBP levels can potentially be used as markers to predict the prognosis of certain cancers.
– A stop-to-sense variant in FEM1B has been observed in hepatocellular carcinoma, suggesting the importance of FEM1B and SLBP levels in tumour progression.
Regulation of SCR by cis- and trans-factors:
– SCR of FEM1B is driven by cis-factors in the 3′UTR immediately downstream of the stop codon.
– In some cases, SCR is positively regulated by trans-factors like microRNAs and proteins.
Cell type-specific expression of FEM1Bx:
– Cell type-specific expression of FEM1Bx was observed, being high in HepG2 cells, low in MDA-MB-231 cells, and undetectable in others.
– The efficiency of SCR based on reporter assays in HeLa cells was determined to be approximately 17%.
Cell Culture:
– HeLa, MDA-MB-231, HEK293, and HepG2 cells were cultured in complete medium at 37°C in a 5% CO2 atmosphere.
– Cell identity was confirmed by STR profiling, and mycoplasma contamination was checked twice a year.
Antibodies and Chemical Reagents:
– Various antibodies and chemical reagents were used, including anti-FEM1B, anti-HA, anti-GFP, anti-SLBP, anti-H2B, anti-H4, and anti-actin.
– Horseradish peroxidase-conjugated secondary antibodies were also used.
RT-PCR:
– Total RNA isolation, cDNA synthesis, semi-quantitative RT-PCR, and quantitative real-time PCR were performed.
– PCR conditions and primer sequences used for RT-PCR were provided.
Deletion of the Proximal Part of the 3’UTR of FEM1B:
– sgRNAs targeting the proximal part of the 3’UTR of FEM1B gene were cloned into a plasmid and transfected into cells.
– Clones were screened for deletion by PCR, confirmed by sequencing.
Colony Formation Assay:
– Cells were seeded at a low density, allowed to grow into colonies, fixed, stained, and counted.
– The assay was used to assess the ability of cells to form colonies.
Cell Proliferation Assay:
– Cells were seeded in a 96-well plate and MTT reagent was used to measure cell proliferation.
– Fold proliferation was calculated based on absorbance measurements.
Cell Cycle Analysis:
– Cells were treated with thymidine to arrest at the G1/S boundary, released into complete medium, fixed, and analyzed for cell cycle distribution.
– The protocol for cell cycle analysis was described in detail.
Conclusion:
– Tissue-specific regulation of SCR process and FEM1B stability was suggested.
– Further investigations are needed to identify the factors responsible for the FEM1B SCR process and its regulation.
Flow Cytometry Analysis:
– Data analysis was performed using the CytExpert software to calculate cell phase percentages.
– Flow cytometry was done using CytoFLEX S from Beckman Coulter.
Western Blotting Procedure:
– Cell lysis was carried out using cell lysis buffer with protease inhibitor for protein extraction.
– Proteins were transferred onto a PVDF membrane for analysis using the Trans-Blot apparatus.
SCR Assays Overview:
– Partial CDS of FEM1B along with ISR was cloned in vectors to study translational readthrough.
– Luminescence and fluorescence assays were performed to measure SCR efficiency.
Mass Spectrometry Data Analysis:
– Raw files from ProteomeXchange were analyzed using MaxQuant software.
– Peptides aligning with FEM1Bx ISR region were identified using an in-house Python script.
Anti-FEM1Bx Antibody Generation:
– Polyclonal antibody specific to C-terminus extended region of FEM1Bx was produced.
– Synthetic peptide dot blot was performed to test antibody specificity.
SHRNA-Mediated Knockdown of FEM1B:
– Cells were electroporated with shRNA constructs and selected with puromycin for knockdown.
– Western blot confirmed the successful knockdown of FEM1B in HepG2 cells.
Generation of FEM1B Knockout HeLa Cells:
– FEM1B gene-editing was performed using the CRISPR-Cas9 system.
– sgRNA sequences were used to target FEM1B gene near its canonical stop codon.
Data Analysis Tools Used:
– ImageJ software was utilized for quantifying band densities in Western blotting.
– MaxQuant software was employed for analyzing mass spectrometry data.
CRISPR Edits Analysis:
– Repair template with UAA→CAA mutation cloned into pmCherry-C1 vector
– Transfection of sgRNA, Cas9, and repair template in HeLa cells followed by colony screening
Dual Fluorescence-based Protein Stability Assay:
– Transfection of DsRed and GFP reporter constructs in HeLa cells
– Quantification of fluorescence intensity using flow cytometry
In Vivo Tumour Experiments:
– Inoculation of HeLa cells in athymic nude mice for tumour progression monitoring
– Tumour volume calculation using Vernier callipers
Sequence Alignment:
– Obtaining 3′UTR nucleotide sequences from NCBI database
– Alignment of sequences using Clustal Omega
Statistical Analysis:
– Calculation of statistical significance using Student’s t-test or ANOVA
– Different tests applied based on experimental design
Acknowledgements:
– Contributors acknowledged for their support and resources
– Funding details and financial support mentioned
Open Access Data Availability:
– Availability disclosure of all data in article and supplementary information
– Link provided for python script used for analysis
References:
– Dynamic regulation of N(6),2′-O-dimethyladenosine in obesity
– Polysome profiling in Plasmodium falciparum
The COP9 signalosome:
– A multi-DUB complex
– Research by Dubiel et al. (2020) in Biomolecules.
Ribosome profiling in Drosophila melanogaster:
– Stop codon readthrough regulation
– Study by Dunn et al. (2013) in Elife.
Programmed translational readthrough:
– Creation of antiangiogenic VEGF-Ax
– Eswarappa et al. (2014) in Cell.
RNA structural element influencing stop codon readthrough:
– Presence of extended 3′ RNA structural element
– Study by Firth et al. (2011) in Nucleic Acids Res.
Fem1b’s role in ubiquitylation and transcriptional activity suppression:
– Gilder et al. (2013) in Biochem. Biophys. Res. Commun.
– Implications for Gli1 regulation.
Novel candidate genes FEM1A and FEM1B in polycystic ovary syndrome:
– Goodarzi et al. (2008) in Hum. Reprod.
– Potential insights into disease mechanisms.
Dual-luciferase reporter system for recoding signals:
– A tool for studying recoding signals
– Grentzmann et al. (1998) in RNA.
BRCA2 functional activity after Homo sapiens divergence:
– Studies by Huang et al. (2022) in Cell Rep.
– Evidence of reduced BRCA2 activity post-divergence.
Role of 3′ UTRs:
– 3′ UTRs play a significant role in regulating gene expression.
– They are involved in post-transcriptional processes.
Comparative Analysis of Cancer Genes:
– A comparison of cancer genes in human and chimpanzee genomes was conducted.
– Insights into evolutionary aspects of cancer genetics were obtained.
mRNA Stop Codon Readthrough:
– Identification and functional characterization of mRNAs exhibiting stop codon readthrough were performed.
– This study was conducted in Arabidopsis thaliana.
Evolutionary Insights from Gorilla Genome:
– The gorilla genome sequence provided insights into hominid evolution.
– Comparative genomic studies were undertaken.
Translational Readthrough Mechanism:
– Peroxisomal lactate dehydrogenase is generated via translational readthrough in mammals.
– This mechanism highlights unique protein synthesis processes.
MicroRNA Pathway Regulation:
– Let-7a-regulated translational readthrough of AGO1 generates a microRNA pathway inhibitor.
– This mechanism adds complexity to microRNA regulation.
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