Biology subjects

Lafranchi, L.

Publications and source records attributed to Lafranchi, L..

3 recordsLinked to original sources

bioRxiv2024

Pooled overexpression screening identifies PIPPI as a novel microprotein involved in the ER stress response

Microproteins encoded by short open reading frames (sORFs) of less than 100 codons have been predicted to constitute a substantial fraction of the eukaryotic proteome. However, relevance and roles of the majority of microproteins remain undefined because only a small fraction of these intriguing cellular players have been in-depth characterized so far. Here we use pooled overexpression screens with a library of 11338 sORFs to overcome the challenge of elucidating which of the thousands of putative translated sORFs are biologically functional. As a proof-of-concept, we performed a phenotypic screen to identify sORFs protecting cells from treatment with the nucleotide analogue 6-thioguanine. With this approach, we identified two cytoprotective microproteins: altDDIT3 and PIPPI. PIPPI is encoded as part of the LC16a core duplicon/Morpheus gene cluster, a highly duplicated region of the human genome, which is undergoing rapid positive selection in primates. Our data show that PIPPI interacts with proteins of the endoplasmic reticulum, including protein disulfide isomerase ERp44. Besides providing mechanistic insights on a new microprotein, this study highlights the power of using pooled overexpression screens to identify functional microproteins.

cell biology↗

bioRxiv2023

A large-scale sORF screen identifies putative microproteins and provides insights into their interaction partners, localisation and function

The human genome contains thousands of potentially coding short open reading frames (sORFs). A growing set of microproteins translated from these sORFs are known to have important cellular functions. However, the majority remains uncharacterised. Thus, larger screens to find functional microproteins have become more vital. Here, we performed a high-throughput CRISPR/Cas9 knock-out screen with a customised library of 11,776 sORFs, curated from literature and databases to identify microproteins essential for cancer cell line growth. 16/17 tested candidates displayed a reproducible knockout phenotype. We selected our top six hits, consisting of 11 to 63 amino acids. Various of these candidates localised to distinct subcellular compartments and the majority showed specific interaction partners. Endogenous tagging demonstrated translation of an sORF in the CENPBD2P pseudogene that bears no resemblance to the CENPBD2P name-giving CENPB DNA binding domains. For two candidates, uORFs in the DSE and NUTF2 genes, the microprotein supplied in trans ameliorated the growth defect of the respective knock-out. RNA-seq analysis revealed however that gene expression changes in the knock-out could only partially be rescued. Overall, we identified various putative microproteins and a microprotein-producing pseudogene that might be involved in cancer cell growth, but also illustrate the limitations and caveats of sORF functional screening and characterisation.

molecular biology↗

bioRxiv2018

Methanomethylophilus alvus Mx1201 provides basis for mutual orthogonal pyrrolysyl tRNA/aminoacyl-tRNA synthetase pairs in mammalian cells

Genetic code expansion via stop codon suppression is a powerful technique for engineering proteins in mammalian cells with site-specifically encoded non-canonical amino acids (ncAAs). Current methods rely on very few available tRNA/aminoacyl-tRNA synthetase pairs orthogonal in mammalian cells, the pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from Methanosarcina mazei (Mma PylRS/PylT) being the most active and versatile to date. We found a previously uncharacterized pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from the human gut archaeon Methanomethylophilus alvus Mx1201 (Mx1201 PylRS/PylT) to be active and orthogonal in mammalian cells. We show that the new PylRS enzyme can be engineered to expand its ncAA substrate spectrum. We find that due to the large evolutionary distance of the two pairs, Mx1201 PylRS/PylT is partially orthogonal to Mma PylRS/PylT. Through rational mutation of Mx1201 PylT, we abolish its non-cognate interaction with Mma PylRS, creating two mutually orthogonal PylRS/PylT pairs. Combined in the same cell, we show that the two pairs can site-selectively introduce two different ncAAs in response to two distinct stop codons. Our work expands the repertoire of mutually orthogonal tools for genetic code expansion in mammalian cells and provides the basis for advanced in vivo protein engineering applications for cell biology and protein production.

synthetic biology↗