Biology subjects

Liss, M.

Publications and source records attributed to Liss, M..

4 recordsLinked to original sources

bioRxiv2026

Splice-Aware Optimization Prevents Pervasive Missplicing of Natural and Synthetic cDNAs

Heterologous gene expression is widely used across biology and medicine, and often relies on codon optimization to increase protein yields. Here we uncover missplicing as a common and largely unrecognized failure mode of heterologous expression. Using systematically designed libraries comprising over 5,000 synthetic reporter genes and natural human cDNAs, we find that the majority of gene variants expressed in a human cell line are at least partially spliced, and in many variants the spliced isoform dominates, reducing protein output or ablating expression entirely. By analysing sequence determinants of expression across multiple human cell lines, we uncover a hierarchical architecture of regulatory control, where GC content establishes baseline mRNA levels, local sequence features influence splicing, and tissue-specific codon adaptation to tRNA pools fine-tunes translation efficiency. These findings enable us to develop predictive models of expression and splicing, benchmark current optimization strategies, and design a splice-aware optimization algorithm that substantially improves transgene performance.

synthetic biology↗

bioRxiv2022

Direct Cell Extraction of Membrane Proteins for Structure-Function Analysis

Membrane proteins are the largest group of therapeutic targets in a variety of disease areas and yet, they remain particularly difficult to investigate. We have developed a novel one-step approach for the incorporation of membrane proteins directly from cells into lipid Salipro nanoparticles. Here, with the pannexin1 channel as a case study, we demonstrate the applicability of this method for structurefunction analysis using SPR and cryo-EM.

biophysics↗

bioRxiv2020

RBM20 phosphorylation on serine/arginine domain is crucial to regulate pre-mRNA splicing and protein shuttling in the heart

Molecular and cellular mechanisms of mutations of splicing factors in heart function are not well understood. The splicing of precursor mRNA is dependent on an essential group of splicing factors containing serine-arginine (SR) domain(s) that are critical for protein-RNA and protein-protein interaction in the spliceosome assembly. Phosphorylation of SR domains plays a key role in splicing control and the distribution of splicing factors in the cell. RNA binding motif 20 (RBM20) is a splicing factor predominantly expressed in muscle tissues with the highest expression level in the heart. However, its phosphorylation status is completely unknown up-to-date. In this study, we identified sixteen amino acid residues that are phosphorylated by middle-down mass spectrometry. Four of them are located in the SR domain, and two out of the four residues, S638 and S640, play an essential role in splicing control and facilitate RBM20 shuttling from the nucleus to the cytoplasm. Re-localization of RBM20 promotes protein aggregation in the cytoplasm. We have also verified that SR-protein kinases (SRPKs), cdc2-like kinases (CLKs) and protein kinase B (PKB or AKT) phosphorylate S638 and S640. Mutations of S638A and S640G reduce RBM20 phosphorylation and disrupt the splicing. Taken together, we determine the phosphorylation status of RBM20 and provide the first evidence that phosphorylation on SR domain is crucial for pre-mRNA splicing and protein trafficking. Our findings reveal a new role of RBM20 via protein shuttling in cardiac function.

cell biology↗

bioRxiv2019

Splicing buffers suboptimal codon usage in human cells

Although multiple studies have addressed the effects of codon usage on gene expression, such studies were typically performed in unspliced model genes. In the human genome, most genes undergo splicing and patterns of codon usage are splicing-dependent: guanine and cytosine (GC) content is highest within single-exon genes and within first exons of multi-exon genes. Intrigued by this observation, we measured the effects of splicing on expression in a panel of synonymous variants of GFP and mKate2 reporter genes that varied in nucleotide composition. We found that splicing promotes the expression of adenine and thymine (AT)-rich variants by increasing their steady-state protein and mRNA levels, in part through promoting cytoplasmic localization of mRNA. Splicing had little or no effect on the expression of GC-rich variants. In the absence of splicing, high GC content at the 5' end, but not at the 3' end of the coding sequence positively correlated with expression. Among endogenous human protein-coding transcripts, GC content has a more positive effect on various expression measures of unspliced, relative to spliced mRNAs. We propose that splicing promotes the expression of AT-rich genes, leading to selective pressure for the retention of introns in the human genome.

synthetic biology↗