Biology subjects

Szpila, M.

Publications and source records attributed to Szpila, M..

4 recordsLinked to original sources

bioRxiv2023

Polyadenylation of mRNAs encoding secreted proteins by TENT5 family of enzymes is essential for gametogenesis in mice.

Cytoplasmic polyadenylation plays a vital role in gametogenesis, however, the participating enzymes and substrates in mammals remain unclear. Using knockout and knock-in mouse models, we describe the essential role of 4 TENT5 poly(A) polymerases in mice fertility and gametogenesis. TENT5B and TENT5C play crucial, but redundant roles in oogenesis, with double knockout of both genes leading to oocyte degeneration. Additionally, TENT5B-GFP knock-in females display gain-of-function infertility effect with multiple chromosomal aberrations in ovulated oocytes. TENT5C and TENT5D both regulate different stages of spermatogenesis, shown by sterility of males with either genes knockout mutation. Finally, Tent5a knockout significantly lowers fertility, although the underlying mechanism is not directly related to gametogenesis. Through Direct RNA sequencing we discovered that TENT5s polyadenylate mRNAs encoding endoplasmic reticulum-targeted proteins essential for gametogenesis. Sequence motives analysis and reporter mRNA assay revealed that the presence of endoplasmic reticulum-leader represents the primary determinant of TENT5-mediated regulation.

developmental biology↗

bioRxiv2023

Recurrent Multiple Myeloma DIS3 alleles arise early but are later counter-selected due to toxicity

DIS3 encodes an essential ribonucleic subunit of the nuclear exosome complex, responsible for degrading RNA in the nucleus. Somatic DIS3 mutations drive translocations in B cells, leading to multiple myeloma (MM). Clinical data analysis reveals that 42% of DIS3 mutations occur at three recurrent residues (D479, D488, and R780). These mutations, deactivating DIS3 exonucleolytic activity, are never homozygous, often appearing as minor subclones in advanced MM. Surprisingly, mutant DIS3 alleles undergo loss-of-heterozygosity, correlating with frequent del(13q) encompassing DIS3. Overexpression of wild-type DIS3 enhances growth and viability of DIS3-mutated MM cells, while CRISPR-mediated knock-out of the mutant variant, followed by longitudinal co-culture, replicates its elimination through counterselection, observed in the natural course of the disease. In mice, the heterozygous DIS3 D479 mutation is embryolethal, confirming its dominant toxic effects. Transcriptome analysis of patients and cell lines reveals specific transcriptional signatures of DIS3 mutations with accumulation of non-coding unstable RNA species and including secondary indications of decreased proliferation. All these signatures are reversible upon mutant DIS3 loss-of-heterozygosity. DIS3 is an intriguing hit-and-run oncogene that drives MM, but is subsequently eliminated during clonal evolution.

molecular biology↗

bioRxiv2022

DIS3L, cytoplasmic exosome catalytic subunit, is essential for development but not cell viability in mice.

Among numerous enzymes involved in RNA decay, processive exoribonucleases are the most prominent group responsible for the degradation of the entire RNA molecules. The role of mammalian cytoplasmic 3-5 exonuclease DIS3L at the organismal level remained unknown. Herein we established knock-in and knock-out mouse models to study DIS3L functions in mice. DIS3L is indeed a subunit of the cytoplasmic exosome complex, which disruption leads to severe embryo degeneration and death in mice soon after implantation. These changes could not be prevented by supplementing extraembryonic tissue with functional DIS3L through the construction of chimeric embryos. Preimplantation Dis3l-/- embryos were unaffected in their morphology and ability to produce functional embryonic stem cells showing that DIS3L is not essential for cell viability. There were also no major changes in the transcriptome level for both embryonic stem cells and blastocysts, as revealed by RNA sequencing experiments. Notably, however, DIS3L knock-out led to inhibition of the global protein synthesis. These results point to the essential role of DIS3L in mRNA quality control pathways crucial for proper protein synthesis during embryo development.

molecular biology↗

bioRxiv2020

Cytoplasmic polyadenylation by TENT5A is required for proper bone formation

Osteoblasts orchestrate bone formation by secreting dense, highly cross-linked type I collagen and other proteins involved in osteogenesis. Mutations in Col11, Col12, or collagen biogenesis factors lead to the human genetic disease, osteogenesis imperfecta (OI). Herein, we show that the TENT5A gene, whose mutation is responsible for poorly characterized type XVIII OI, encodes an active cytoplasmic poly(A) polymerase regulating osteogenesis. TENT5A is induced during osteoblast differentiation and TENT5A KO osteoblasts are defective in mineralization. The TENT5A KO mouse recapitulates OI disease symptoms such as bone fragility and hypomineralization. Direct RNA sequencing revealed that TENT5A polyadenylates and increases expression of Col11 and Col12 RNAs, as well as those of other genes mutated in OI, resulting in lower production and improper folding of collagen chains. Thus, we have identified the specific pathomechanism of XVIII OI and report for the first time a biologically relevant post-transcriptional regulator of collagen production. We further postulate that TENT5A, possibly together with its paralogue TENT5C, is responsible for the wave of cytoplasmic polyadenylation of mRNAs encoding secreted proteins occurring during bone mineralization.

genetics↗