Biology subjects

Mroczek, S.

Publications and source records attributed to Mroczek, S..

10 recordsLinked to original sources

bioRxiv2025

Post-transcriptional control of enamel matrix mineralization by TENT5A-mediated cytoplasmic polyadenylation

Tooth enamel formation, or amelogenesis, is a complex biomineralization process regulated by ameloblasts cells. These cells secrete enamel matrix proteins (EMPs), including Amelogenin (AMELX) and Ameloblastin (AMBN), which are essential for hydroxyapatite crystal deposition during enamel mineralization. Precise regulation of this process ensures the mechanical and chemical strength of dental enamel. Using Tent5a knock-out (KO) mouse, we demonstrated that TENT5A, a non-canonical poly(A) polymerase is crucial for the stability, translation, and secretion of EMP mRNAs, particularly AMELX, during amelogenesis. TENT5A-deficient mice exhibited Amelogenesis imperfecta, characterized by enamel hypomineralization, reduced thickness, and disrupted ultrastructure, as revealed by micro-computed tomography. Through nanopore direct mRNA sequencing, we identified that TENT5A polyadenylates Amelx and other mRNAs encoding secreted proteins, enhancing their stability and translation in the endoplasmic reticulum. Moreover, loss of TENT5A altered AMELX secretion and extracellular self-assembly, impairing matrix organization and hydroxyapatite deposition. This study highlights the role of TENT5A in post-transcriptional regulation during enamel formation, demonstrating its importance in enamel homeostasis. TeaserTENT5A adds poly(A) tails to specific mRNAs in enamel, enhancing their expression, which is crucial for dental mineralization.

developmental biology↗

bioRxiv2025

PolyA tail segmentation improves the stability of the template DNA and increases the translatability of in vitro transcribed mRNA

PolyA tail regulates mRNA localization, stability, and translation. PolyA length affects the durability and translational activity of both endogenous and exogenously delivered mRNAs. However, long polyA stretches can undergo recombination during amplification in bacterial plasmids, impairing the production of in vitro transcribed (IVT) mRNA with long polyAs. PolyA tail segmentation with heteronucleotide spacers has recently emerged as a solution. Here, we developed segmented polyA patterns that stabilize the sequence during DNA amplification and enhance mRNA translation. We designed 15 novel genetically modified polyA variants, differing in the length, placement, and frequency of spacers, and the overall length (from [~]120 to [~]200 nucleotides). We evaluated their stability in DNA plasmids and homogeneity, translational activity, and durability in cell culture of the resulting mRNAs, comparing them to A90 tail and other known solutions, including those from existing mRNA vaccines. Selected sequences were validated in vivo. Surprisingly, we found that even frequent heteronucleotide insertions produce functional polyA tails. The most notable enhancements in protein production were observed for a segmented tail exceeding 200 nt in length [A30(CA15)11; up to 6-fold compared to mRNA with A90 tails]. Our findings extend the scope of possible polyA modification strategies, offering new possibilities for advancing mRNA therapeutics.

molecular biology↗

bioRxiv2024

The polyadenylation landscape after in vivo long-term potentiation in the rat brain

Local protein synthesis in neurons is vital for synaptic maintenance and plasticity, yet the regulatory mechanisms, particularly cytoplasmic polyadenylation, are not fully understood. This study employed nanopore sequencing to examine transcriptomic responses in rat hippocampi during in vivo long-term potentiation (LTP) and in synaptoneurosomes after in vitro stimulation. Our long-read transcriptomic dataset allows for detailed analysis of mRNA 3'-ends, poly(A) tail lengths, and nucleotide composition. We observed dynamic shifts in polyadenylation site preference post-LTP induction, with significant poly(A) tail lengthening restricted to transcriptionally induced mRNAs. The poly(A) tails of these genes showed increased non-adenosine abundance. In synaptoneurosomes, chemical stimulation led to shortening of poly(A) tails on preexisting mRNAs, indicating translation-induced deadenylation. Additionally, we discovered a group of neuronal transcripts with poly(A) tails abundant in non-adenosine residues. These tails are semi-templated and derived from extremely adenosine-rich 3'UTRs. This study provides a comprehensive overview of mRNA 3'-end dynamics during LTP, offering insights into post-transcriptional regulation following synaptic activation of plasticity in neurons.

neuroscience↗

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↗

bioRxiv2023

mRNA decay can be uncoupled from deadenylation during stress response

The polyadenosine tail (pA-tail) regulates mRNA nuclear export, stability, and translatability. Based on reporter constructs, the prevailing model suggests that pA-tail removal mediated by Ccr4-NOT or PAN2/3 deadenylases is required for mRNA decapping and degradation. Here, we use direct RNA sequencing to track mRNA deadenylation and decay at steady-state and in stress conditions to show a global correlation between deadenylation and decay. Interestingly, codon optimality, previously postulated to dictate mRNA stability, only strongly affects decay of conserved and abundant transcripts, such as coding for ribosomal protein subunits. Degradation of those mRNAs is also accelerated in response to stress. Still, the in-depth analysis revealed that deadenylation is a factor that contributes to degradation but is not indispensable for decapping. We further demonstrate that deadenylation is the fastest for newly made tails depending on polyA-binding protein Pab1. Unexpectedly, decapping initiates on mRNAs of pA-tails of 20-35 adenosines presumably bound by Pab1.

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↗

bioRxiv2022

SARS-CoV-2 mRNA vaccine is re-adenylated in vivo, enhancing antigen production and immune response

Though mRNA vaccines against COVID-19 have revolutionized vaccinology and have been administered in billions of doses, we know incredibly little about how mRNA vaccines are metabolized in vivo. Here we implemented enhanced nanopore Direct RNA sequencing (eDRS), to enable the analysis of single Modernas mRNA-1273 molecules, giving in vivo information about the sequence and poly(A) tails. We show that mRNA-1273, with all uridines replaced by N1-methylpseudouridine (m{Psi}), is terminated by a long poly(A) tail (~100 nucleotides) followed by an m{Psi}Cm{Psi}AG sequence. In model cell lines, mRNA-1273 is swiftly degraded in a process initiated by the removal of m{Psi}Cm{Psi}AG, followed by CCR4-NOT-mediated deadenylation. In contrast, intramuscularly inoculated mRNA-1273 undergoes more complex modifications. Notably, mRNA-1273 molecules are re-adenylated after m{Psi}Cm{Psi}AG removal. Detailed analysis of immune cells involved in antigen production revealed that in macrophages, after m{Psi}Cm{Psi}AG removal, vaccine mRNA is very efficiently re-adenylated, and poly(A) tails can reach up to 200A. In contrast, in dendritic cells, vaccine mRNA undergoes slow deadenylation-dependent decay. We further demonstrate that enhancement of mRNA stability in macrophages is mediated by TENT5 poly(A) polymerases, whose expression is induced by the vaccine itself. Lack of TENT5-mediated re-adenylation results in lower antigen production and severely compromises specific immunoglobulin production following vaccination. Together, our findings provide an unexpected principle for the high efficacy of mRNA vaccines and open new possibilities for their improvement. They also emphasize that, in addition to targeting a protein of interest, the design of mRNA therapeutics should be customized to its cellular destination.

immunology↗

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↗

bioRxiv2019

B-cell humoral response and differentiation is regulated by non-canonical poly(A) polymerase TENT5C

TENT5C is a non-canonical cytoplasmic poly(A) polymerase (ncPAP) upregulated in activated B cells and suppressing their proliferation. Herein we measured the global distribution of poly(A) tail lengths in responsive B cells using a modified Nanopore direct RNA-sequencing approach and revealed that TENT5C polyadenylates immunoglobulin mRNAs regulating their steady-state levels. Consequently, TENT5C deficient B cells secrete less antibodies and KO mice have diminished gamma globulin concentrations despite the increased number of CD138high plasma cells as a consequence of accelerated differentiation. TENT5C is explicitly upregulated in differentiating plasma cells by innate signaling. Importantly, TENT5C deficiency in B lymphocytes impairs the capacity of the secretory pathway through the reduction of ER volume and downregulation of unfolded protein response.\n\nOur findings define the role of the TENT5C enzyme in B cell physiology and discover the first ncPAP engaged in the regulation of immunoglobulin mRNA poly(A) tails, thus serving as a regulator of humoral immunity.

immunology↗