Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2024.02.04.578419

Functional Analysis of the Zinc Finger Modules of the S. cerevisiae Splicing Factor Luc7

Carrocci, T. J.·DeMario, S. M.·He, K.·Zeps, N. J.·Harkner, C.·CHANFREAU, G. F.·Hoskins, A.
View original source ↗Export metadata ↓

Abstract

Identification of splice sites is a critical step in pre-mRNA splicing since definition of the exon/intron boundaries controls what nucleotides are incorporated into mature mRNAs. The intron boundary with the upstream exon is initially identified through interactions with the U1 snRNP. This involves both base pairing between the U1 snRNA and the pre-mRNA as well as snRNP proteins interacting with the 5 splice site/snRNA duplex. In yeast, this duplex is buttressed by two conserved protein factors, Yhc1 and Luc7. Luc7 has three human paralogs (LUC7L, LUC7L2, and LUC7L3) which play roles in alternative splicing. What domains of these paralogs promote splicing at particular sites is not yet clear. Here, we humanized the zinc finger domains of the yeast Luc7 protein in order to understand their roles in splice site selection using reporter assays, transcriptome analysis, and genetic interactions. While we were unable to determine a function for the first zinc finger domain, humanization of the second zinc finger domain to mirror that found in LUC7L or LUC7L2 resulted in altered usage of nonconsensus 5 splice sites. In contrast, the corresponding zinc finger domain of LUC7L3 could not support yeast viability. Further, humanization of Luc7 can suppress mutation of the ATPase Prp28, which is involved in U1 release and exchange for U6 at the 5 splice site. Our work reveals a role for the second zinc finger of Luc7 in splice site selection and suggests that different zinc finger domains may have different ATPase requirements for release by Prp28.

Source connections

Explore related subjects

biochemistryPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Carrocci, T. J., DeMario, S. M., He, K., Zeps, N. J., Harkner, C., CHANFREAU, G. F., Hoskins, A.. 2024-02-04. Functional Analysis of the Zinc Finger Modules of the S. cerevisiae Splicing Factor Luc7. https://doi.org/10.1101/2024.02.04.578419

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Impacts of Environmental Nickel and Chromium Exposure on the Blood Proteome of House Sparrows (Passer domesticus) in New Caledonia

New Caledonia holds some of the world's largest ultramafic mineral deposits, and nickel (Ni) smelting operations have created a gradient of Ni and chromium (Cr) contamination across the archipelago. Molecular impacts of Ni and Cr toxicity on wildlife remain largely unexplored. Here, we used the house sparrow (Passer domesticus) as a sentinel species to characterise the blood proteomic response to Ni and Cr exposure near the Doniambo smelter in Noumea. Blood was collected from 52 free-living sparrows across 18 sites classified by soil metal concentration. Plasma and blood cell pellet fractions were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Plasma proteomics identified histidine-rich glycoprotein (HRG) as the most prominent responder, with approximately 2.1-fold elevation in exposed relative to low-exposure birds. HRG contains a conserved histidine-rich region that binds cations including divalent nickel, and paired plasma-liver analysis confirmed hepatic-driven synthesis. Blood cell pellet proteomics revealed that Ni-Cr co-exposure predominantly disrupts the nuclear compartment: histone depletion indicated chromatin collapse, NUP153 dysregulation impaired nuclear pore function, and VRK1/TRIP13 upregulation suggested DNA damage response activation coupled with mitotic checkpoint override. Concurrent RNA processing collapse (DDX17, DHX15, RBMX, ALYREF) and changes in telomere-associated proteins (TERF2IP, hnRNPA1 and VRK1) implicated disrupted genome maintenance. These findings identify HRG as a candidate plasma biomarker of Ni-Cr exposure and reveal a coordinated nuclear toxicity signature linking heavy metal contamination to DNA damage and epigenetic disruption in a free-living vertebrate.

biochemistry↗
bioRxiv2026

Programmable amide bond formation using de novo ligases

Amide bond formation is of central importance in the chemical industry, underpinning the synthesis of small bioactive molecules, macrocycles, peptides and their conjugates. Conventional methods of amide synthesis rely on stoichiometric coupling agents and costly protecting group manipulations. While biocatalysis can alleviate some of these challenges, enzymes typically operate across a narrow range of substrates and their adaptation to new coupling partners requires extensive engineering. Here we show that de novo amide ligases featuring a cysteine catalytic triad can be programmed computationally to operate across diverse substrate classes with high selectivity. We first designed complementary peptide ligases to selectively functionalise either the N-terminus or a single lysine residue of a polypeptide sequence containing 36 competing lysines. Crystal structures of both ligases show excellent agreement with the designs, with structural modelling suggesting that amidation outcompetes hydrolysis because the catalytically competent geometry forms only upon acceptor binding. We then extended our approach to the synthesis of small bioactive molecules by designing stereoselective ligases that mediate a key amide bonding forming step in the synthesis of the preventative HIV therapeutic lenacapavir. These results establish de novo enzyme design as a programmable platform for achieving selective amide bond formations across a wide range of molecular targets.

biochemistry↗
bioRxiv2026

De novo design of ligases for programmable site-specific protein ubiquitylation

Protein ubiquitylation regulates numerous cellular processes, but difficulties in generating site-specifically ubiquitylated proteins limit our understanding of individual modification events. In cells, ubiquitylation is mediated by an E1-E2-E3 enzyme cascade, with substrate recognition largely determined by more than 600 human E3 ligases, making it challenging to redirect ubiquitin transfer to a chosen lysine. Here we use RFdiffusion3 to design ubiquitin ligases from scratch that use a cysteine-based catalytic triad to conjugate an activated ubiquitin donor to a selected lysine in a chosen target substrate. Of 48 tested designs, 35 ubiquitylated a model peptide, with the most active ligases achieving near-quantitative ubiquitin transfer. We next challenged the approach with the homotrimeric sliding clamp proliferating cell nuclear antigen (PCNA), whose ubiquitylation at lysine (K) 164 governs DNA damage tolerance while displaying 45 off-target lysines for ubiquitination. Screening of 48 designs delivered a site-selective ligase. Incorporation of a single activity-increasing substitution and coupling to E1-dependent donor activation resulted in 90% conversion from unactivated ubiquitin, delivering higher rates and superior selectivity compared to a natural promiscuous E2. Using this cascade, we transferred di-, tri- and tetraubiquitin chains of defined length to PCNA K164 through a native isopeptide bond. These results establish de novo enzyme design as a route towards programmable, site-specific protein ubiquitylation. That a single de novo designed protein can bind both an activated ubiquitin donor and a globular target, and use a proficient catalytic machinery to selectively transfer ubiquitin to one lysine among 45, illustrates the level of functional complexity now within reach of de novo enzyme design.

biochemistry↗