Biology subjects

Futcher, B.

Publications and source records attributed to Futcher, B..

9 recordsLinked to original sources

bioRxiv2025

Cln3 can work independently of Whi5 on the cell size for Start in yeast

In yeast, cells commit to division at "Start" in G1 phase. The critical cell size for Start is regulated in part by the size-control genes CLN3 and WHI5. Cln3 is a G1 cyclin that activates Start by activating the Cdc28 cyclin dependent kinase. Null cln3 mutants have large cells, because they lack an activator, while hyperactive mutants have small cells. Whi5 inhibits Start. Null whi5 mutants have small cells, while hyperactive mutants have large cells. Previous studies suggested that CLN3 and WHI5 are in a linear, dependent pathway, in which Cln3 acts inhibits Whi5, possibly by phosphorylation. We compared isogenic WT, cln3, whi5, and cln3 whi5 mutants, and find that whi5 is not epistatic, or not fully epistatic, to CLN3--that is, CLN3 can work independently of WHI5. The cln3 and whi5 deletions have off-setting phenotypes, such that the double mutant is nearly wild-type in cell size, and has only the mildest phenotype, with well-controlled cell size, despite entirely lacking two major cell size regulators. Growth rates were also measured. Some mutants were larger and some mutants smaller than wild-type, but the wild-type had the fastest doubling time, consistent with the idea that wild-type size is optimal for growth rate.

genetics↗

bioRxiv2025

A Trizol-Beadbeater RNA extraction method for yeast, and comparison to Novogene RNA extraction

58 samples of S. cerevisiae were sent to the Novogene Corporation for the intended purpose of RNA extraction, cDNA preparation, and sequencing. RNA was extracted by Novogene. However, yields of total RNA were about 1%, perhaps 1.25%, of the theoretically expected amount, which seemed low. Therefore, we extracted RNA locally using Trizol and bead-beating from a second, identical aliquot of 55 of the samples sent to Novogene. This local extraction gave yields that were on average 40% of the theoretical expectation, about 40 fold higher than the yields obtained by Novogene.

molecular biology↗

bioRxiv2025

Eusocial Reproduction Selects for Longevity

Animals such as bees, ants, wasps, termites, and naked mole-rats live in colonies in which a single queen is the only female reproductive, an arrangement known as eusociality. Eusocial animals are known for their remarkably long lifespans. It has been argued that longevity becomes selected when queens are shielded from "external mortality". While such protection may contribute, we find a deeper reason: the eusocial reproduction strategy itself inherently creates selection for long lifespans. Lifespans typically reflect two processes: the baseline risk of death and the rate at which this risk increases with age. Each is a parameter in the Gompertz mortality equation. We show that the mathematical properties of eusocial reproduction lead to slowly-growing, older populations where selection acts more strongly on the rate at which risk increases than on the baseline risk. In addition, we show that channeling reproduction through a single female also selects for longevity, which we term the "queen effect". Thus, the dynamics of eusocial reproduction select for longer lifespan. More broadly, these results show that reproductive structure and population growth dynamics can fundamentally shape selection on lifespan, with implications outside eusocial systems as well.

evolutionary biology↗

bioRxiv2023

The yeast ribosomal protein Rpl1b is not required for respiration

Previously, Segev and Gerst found that mutants in any of the four ribosomal protein genes rpl1b, rpl2b, rps11a, or rps26b had a petite phenotype--i.e., the mutants were deficient in respiration. Strikingly, mutants of their paralogs rpl1a, rpl2a, rps11b, and rps26a were grande--i.e., competent for respiration. It is remarkable that these paralogs should have opposite phenotypes, because three of the paralog pairs (Rpl1a/Rpl1b, Rpl2a/Rpl2b, Rps11a/Rps11b) are 100% identical to each other in amino acid sequence, while Rps26a and Rps26b differ in 2 amino acids out of 119. However, while attempting to use this paralog-specific petite phenotype in an unrelated experiment, I found that the rpl1b, rpl2b, rps11a, and rps26b deletion mutants are competent for respiration, contrary to Segev and Gerst.

cell biology↗

bioRxiv2023

Gene recoding by synonymous mutations creates promiscuous intragenic transcription initiation in mycobacteria

Each genome encodes some codons more frequently than their synonyms (codon usage bias), but codons are also arranged more frequently into specific pairs (codon pair bias). Recoding viral genomes and yeast or bacterial genes with non-optimal codon pairs has been shown to decrease gene expression. Gene expression is thus importantly regulated not only by the use of particular codons but by their proper juxtaposition. We therefore hypothesized that non-optimal codon pairing could likewise attenuate Mtb genes. We explored the role of codon pair bias by recoding Mtb genes (rpoB, mmpL3, ndh) and assessing their expression in the closely related and tractable model organism M. smegmatis. To our surprise, recoding caused the expression of multiple smaller protein isoforms from all three genes. We confirmed that these smaller proteins were not due to protein degradation, but instead issued from new transcription initiation sites positioned within the open reading frame. New transcripts gave rise to intragenic translation initiation sites, which in turn led to the expression of smaller proteins. We next identified the nucleotide changes associated with these new sites of transcription and translation. Our results demonstrated that apparently benign, synonymous changes can drastically alter gene expression in mycobacteria. More generally, our work expands our understanding of the codon-level parameters that control translation and transcription initiation. IMPORTANCEMycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the deadliest infectious diseases worldwide. Previous studies have established that synonymous recoding to introduce rare codon pairings can attenuate viral pathogens. We hypothesized that non-optimal codon pairing could be an effective strategy for attenuating gene expression to create a live vaccine for Mtb. We instead discovered that these synonymous changes enabled the transcription of functional mRNA that initiated in the middle of the open reading frame and from which many smaller protein products were expressed. To our knowledge, this is the first report that synonymous recoding of a gene in any organism can create or induce intragenic transcription start sites.

molecular biology↗

bioRxiv2022

Slow Translation of the 5' ends of genes may be a non-adaptive consequence of their rapid evolutionary turn-over and slow loss of rare codons.

Previously, Tuller et al. found that the first 30 to 50 codons of the genes of yeast and other eukaryotes are slightly enriched for rare codons, so are presumably translated somewhat slowly. They argued, based on informatics, that this initial slow translation "ramp" was adaptive; and increased efficiency of translation by queuing ribosomes to prevent collisions. Today, the translational speeds of different codons are known, and indeed rare codons are translated slowly. We re-examined the slow translation ramp. We confirm the finding that 5 regions are enriched for rare codons. However, we also find that the 5 ends of yeast genes are poorly conserved in evolution, suggesting that they are unstable and turn over relatively rapidly. When a new 5 end forms de novo, it is likely to include codons that would otherwise be rare. Because evolution has had a relatively short time to select against these codons, 5 ends are typically slightly enriched for rare, slow codons. Opposite to the expectation of Tuller et al., we show by direct experiment that genes with slowly translated codons at the 5 end are expressed relatively poorly, and substituting faster codons improves expression. Further informatic studies suggest that for natural genes, slow 5 ends are correlated with poor gene expression, opposite to the expectation of Tuller et al. Thus we conclude that slow 5 translation is a "spandrel"; it is a non-adaptive consequence of something else, in this case the turnover of 5 ends in evolution, and it does not improve translation. HighlightThe 5 ends of yeast genes are unstable over evolutionary time, enriching for rare codons, slowing translation; slow initial translation does not enhance expression.

molecular biology↗

bioRxiv2022

A Statistical Detector for Ribosomal Frameshifts and Dual Encodings based on Ribosome Profiling

During protein synthesis, the ribosome shifts along the messenger RNA (mRNA) by exactly three nucleotides for each amino acid added to the protein being translated. However, in special cases, the sequence of the mRNA somehow induces the ribosome to shift forward by either two or four nucleotides. This shifts the "reading frame" in which the mRNA is translated, and gives rise to an otherwise unexpected protein. Such "programmed frameshifts" are well-known in viruses, including coronavirus, and a few cases of programmed frameshifting are also known in cellular genes. However, there is no good way, either experimental or informatic, to identify novel cases of programmed frameshifting. Thus it is possible that substantial numbers of cellular proteins generated by programmed frameshifting in human and other organisms remain unknown. Here, we build on prior work observing that data from ribosome profiling can be analyzed for anomalies in mRNA reading frame periodicity to identify putative programmed frameshifts. We develop a statistical framework to identify all likely (even for very low frameshifting rates) frameshift positions in a genome. We also develop a frameshift simulator for ribosome profiling data to verify our algorithm. We show high sensitivity of prediction on the simulated data, retrieving 97.4% of the simulated frameshifts. Furthermore, our method found all three of the known yeast genes with programmed frameshifts. We list several hundred yeast genes that may contain +1 or -1 frameshifts. Our results suggest there could be a large number of un-annotated alternative proteins in the yeast genome generated by programmed frameshifting. This motivates further study and parallel investigations in the human genome. Frameshift Detector algorithms and instructions can be accessed in Github: https://github.com/ayurovsky/Frame-Shift-Detector.

bioinformatics↗

bioRxiv2021

Whi5 and Stb1 define redundant pathways through which the G1 cyclin Cln3 promotes cell division

Withdrawal StatementThe authors have withdrawn their manuscript owing to a defect in the WHI5* STB1* strain used in Fig. 3, Fig. 4, and Table 2. In this strain, for reasons still unknown to us, the GAL-CLN3 gene is expressed moderately well even in the absence of galactose, and is only slightly induced upon the addition of galactose. Since CLN3 is not being substantially induced in the experiments of Fig. 3 and Fig. 4 in this WHI5* STB1* strain, no conclusion can be drawn as to whether there is or is not signaling from CLN3 to Start in a WHI5* STB1* mutant. Results in the manuscript not involving this strain appear to be correct. If you have any questions, please contact the corresponding author.

genetics↗

bioRxiv2018

Extensive recoding of dengue virus type 2 specifically reduces replication in primate cells without gain-of-function in Aedes aegypti mosquitoes

Dengue virus (DENV), an arthropod-borne (\"arbovirus\") virus causing a range of human maladies ranging from self-limiting dengue fever to the life-threatening dengue shock syndrome, proliferates well in two different taxa of the Animal Kingdom, mosquitoes and primates. Unexpectedly, mosquitoes and primates have distinct preferences when expressing their genes by translation, e.g. members of these taxa show taxonomic group-specific intolerance to certain codon pairs. This is called \"codon pair bias\". By necessity, arboviruses evolved to delicately balance this fundamental difference in their ORFs. Using the mosquito-borne human pathogen DENV we have undone the evolutionarily conserved genomic balance in its ORF sequence and specifically shifted the encoding preference away from primates. However, this recoding of DENV raised concerns of gain-of-function, namely whether recoding could inadvertently increase fitness for replication in the arthropod vector. Using mosquito cell cultures and two strains of Aedes aegypti we did not observe any increase in fitness in DENV2 variants codon pair deoptimized for humans. This ability to disrupt and control an arboviruss host preference has great promise towards developing the next generation of synthetic vaccines not only for DENV but for other emerging arboviral pathogens such as chikungunya virus and Zika virus.

microbiology↗