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Biomacromolecules Sep 2023While biomaterials have become indispensable for a wide range of tissue repair strategies, second removal procedures oftentimes needed in the case of non-bio-based and...
While biomaterials have become indispensable for a wide range of tissue repair strategies, second removal procedures oftentimes needed in the case of non-bio-based and non-bioresorbable scaffolds are associated with significant drawbacks not only for the patient, including the risk of infection, impaired healing, or tissue damage, but also for the healthcare system in terms of cost and resources. New biopolymers are increasingly being investigated in the field of tissue regeneration, but their widespread use is still hampered by limitations regarding mechanical, biological, and functional performance when compared to traditional materials. Therefore, a common strategy to tune and broaden the final properties of biopolymers is through the effect of different reinforcing agents. This research work focused on the fabrication and characterization of a bio-based and bioresorbable composite material obtained by compounding a poly(3-hydroxybutyrate--3-hydroxyhexanoate) (PHBH) matrix with acetylated cellulose nanocrystals (CNCs). The developed biocomposite was further processed to obtain three-dimensional scaffolds by additive manufacturing (AM). The 3D printability of the PHBH-CNC biocomposites was demonstrated by realizing different scaffold geometries, and the results of in vitro cell viability studies provided a clear indication of the cytocompatibility of the biocomposites. Moreover, the CNC content proved to be an important parameter in tuning the different functional properties of the scaffolds. It was demonstrated that the water affinity, surface roughness, and in vitro degradability rate of biocomposites increase with increasing CNC content. Therefore, this tailoring effect of CNC can expand the potential field of use of the PHBH biopolymer, making it an attractive candidate for a variety of tissue engineering applications.
Topics: Humans; Cellulose; Poly A; Hydroxybutyrates; Printing, Three-Dimensional
PubMed: 37589321
DOI: 10.1021/acs.biomac.3c00263 -
International Journal of Molecular... Mar 2021Most protein-coding genes in eukaryotes possess at least two poly(A) sites, and alternative polyadenylation is considered a contributing factor to transcriptomic and... (Review)
Review
Most protein-coding genes in eukaryotes possess at least two poly(A) sites, and alternative polyadenylation is considered a contributing factor to transcriptomic and proteomic diversity. Following transcription, a nascent RNA usually undergoes capping, splicing, cleavage, and polyadenylation, resulting in a mature messenger RNA (mRNA); however, increasing evidence suggests that transcription and RNA processing are coupled. Plants, which must produce rapid responses to environmental changes because of their limited mobility, exhibit such coupling. In this review, we summarize recent advances in our understanding of the coupling of transcription with RNA processing in plants, and we describe the possible spatial environment and important proteins involved. Moreover, we describe how liquid-liquid phase separation, mediated by the C-terminal domain of RNA polymerase II and RNA processing factors with intrinsically disordered regions, enables efficient co-transcriptional mRNA processing in plants.
Topics: Gene Expression Regulation, Plant; Polyadenylation; RNA, Messenger; RNA, Plant; Transcription, Genetic
PubMed: 33804866
DOI: 10.3390/ijms22073300 -
Nature Communications Nov 2023The genomic distribution of cleavage and polyadenylation (polyA) sites should be co-evolutionally optimized with the local gene structure. Otherwise, spurious...
The genomic distribution of cleavage and polyadenylation (polyA) sites should be co-evolutionally optimized with the local gene structure. Otherwise, spurious polyadenylation can cause premature transcription termination and generate aberrant proteins. To obtain mechanistic insights into polyA site optimization across the human genome, we develop deep/machine learning models to identify genome-wide putative polyA sites at unprecedented nucleotide-level resolution and calculate their strength and usage in the genomic context. Our models quantitatively measure position-specific motif importance and their crosstalk in polyA site formation and cleavage heterogeneity. The intronic site expression is governed by the surrounding splicing landscape. The usage of alternative polyA sites in terminal exons is modulated by their relative locations and distance to downstream genes. Finally, we apply our models to reveal thousands of disease- and trait-associated genetic variants altering polyadenylation activity. Altogether, our models represent a valuable resource to dissect molecular mechanisms mediating genome-wide polyA site expression and characterize their functional roles in human diseases.
Topics: Humans; Polyadenylation; Nucleotides; Deep Learning; Transcription, Genetic; Genome, Human
PubMed: 37968271
DOI: 10.1038/s41467-023-43266-3 -
BMC Bioinformatics Sep 2022The eukaryotic genome is capable of producing multiple isoforms from a gene by alternative polyadenylation (APA) during pre-mRNA processing. APA in the 3'-untranslated...
BACKGROUND
The eukaryotic genome is capable of producing multiple isoforms from a gene by alternative polyadenylation (APA) during pre-mRNA processing. APA in the 3'-untranslated region (3'-UTR) of mRNA produces transcripts with shorter or longer 3'-UTR. Often, 3'-UTR serves as a binding platform for microRNAs and RNA-binding proteins, which affect the fate of the mRNA transcript. Thus, 3'-UTR APA is known to modulate translation and provides a mean to regulate gene expression at the post-transcriptional level. Current bioinformatics pipelines have limited capability in profiling 3'-UTR APA events due to incomplete annotations and a low-resolution analyzing power: widely available bioinformatics pipelines do not reference actionable polyadenylation (cleavage) sites but simulate 3'-UTR APA only using RNA-seq read coverage, causing false positive identifications. To overcome these limitations, we developed APA-Scan, a robust program that identifies 3'-UTR APA events and visualizes the RNA-seq short-read coverage with gene annotations.
METHODS
APA-Scan utilizes either predicted or experimentally validated actionable polyadenylation signals as a reference for polyadenylation sites and calculates the quantity of long and short 3'-UTR transcripts in the RNA-seq data. APA-Scan works in three major steps: (i) calculate the read coverage of the 3'-UTR regions of genes; (ii) identify the potential APA sites and evaluate the significance of the events among two biological conditions; (iii) graphical representation of user specific event with 3'-UTR annotation and read coverage on the 3'-UTR regions. APA-Scan is implemented in Python3. Source code and a comprehensive user's manual are freely available at https://github.com/compbiolabucf/APA-Scan .
RESULT
APA-Scan was applied to both simulated and real RNA-seq datasets and compared with two widely used baselines DaPars and APAtrap. In simulation APA-Scan significantly improved the accuracy of 3'-UTR APA identification compared to the other baselines. The performance of APA-Scan was also validated by 3'-end-seq data and qPCR on mouse embryonic fibroblast cells. The experiments confirm that APA-Scan can detect unannotated 3'-UTR APA events and improve genome annotation.
CONCLUSION
APA-Scan is a comprehensive computational pipeline to detect transcriptome-wide 3'-UTR APA events. The pipeline integrates both RNA-seq and 3'-end-seq data information and can efficiently identify the significant events with a high-resolution short reads coverage plots.
Topics: 3' Untranslated Regions; Animals; Fibroblasts; Mice; MicroRNAs; Polyadenylation; Protein Isoforms; RNA Precursors; RNA, Messenger; RNA-Seq
PubMed: 36171568
DOI: 10.1186/s12859-022-04939-w -
Development (Cambridge, England) Jun 2022As one of the post-transcriptional regulatory mechanisms, uncoupling of transcription and translation plays an essential role in development and adulthood physiology....
As one of the post-transcriptional regulatory mechanisms, uncoupling of transcription and translation plays an essential role in development and adulthood physiology. However, it remains elusive how thousands of mRNAs get translationally silenced while stability is maintained for hours or even days before translation. In addition to oocytes and neurons, developing spermatids display significant uncoupling of transcription and translation for delayed translation. Therefore, spermiogenesis represents an excellent in vivo model for investigating the mechanism underlying uncoupled transcription and translation. Through full-length poly(A) deep sequencing, we discovered dynamic changes in poly(A) length through deadenylation and re-polyadenylation. Deadenylation appeared to be mediated by microRNAs (miRNAs), and transcripts with shorter poly(A) tails tend to be sequestered into ribonucleoprotein (RNP) granules for translational repression and stabilization. In contrast, re-polyadenylation might allow for translocation of the translationally repressed transcripts from RNP granules to polysomes. Overall, our data suggest that miRNA-dependent poly(A) length control represents a previously unreported mechanism underlying uncoupled translation and transcription in haploid male mouse germ cells.
Topics: Animals; Haploidy; Male; Mice; MicroRNAs; Poly A; Protein Biosynthesis; RNA, Messenger; Spermatids
PubMed: 35588208
DOI: 10.1242/dev.199573 -
International Journal of Molecular... Apr 2023Polyadenylation (polyA) defines the 3' boundary of a transcript's genetic information. Its position can vary and alternative polyadenylation (APA) transcripts can exist...
Polyadenylation (polyA) defines the 3' boundary of a transcript's genetic information. Its position can vary and alternative polyadenylation (APA) transcripts can exist for a gene. This causes variance in 3' regulatory domains and can affect coding sequence if intronic events occur. The distribution of polyA sites on articular chondrocyte transcripts has not been studied so we aimed to define their transcriptome-wide location in age-matched healthy and osteoarthritic knee articular cartilage. Total RNA was isolated from frozen tissue samples and analysed using the QuantSeq-Reverse 3' RNA sequencing approach, where each read runs 3' to 5' from within the polyA tail into the transcript and contains a distinct polyA site. Differential expression of transcripts was significant altered between healthy and osteoarthritic samples with enrichment for functionalities that were strongly associated with joint pathology. Subsequent examination of polyA site data allowed us to define the extent of site usage across all the samples. When comparing healthy and osteoarthritic samples, we found that differential use of polyadenylation sites was modest. However, in the genes affected, there was potential for the APA to have functional relevance. We have characterised the polyadenylation landscape of human knee articular chondrocytes and conclude that osteoarthritis does not elicit a widespread change in their polyadenylation site usage. This finding differentiates knee osteoarthritis from pathologies such as cancer where APA is more commonly observed.
Topics: Humans; Polyadenylation; Cartilage, Articular; Transcriptome; Osteoarthritis, Knee; Sequence Analysis, RNA; RNA
PubMed: 37047586
DOI: 10.3390/ijms24076611 -
Molecular Biology and Evolution Jun 2023Differential regulation of gene expression has produced the astonishing diversity of life on Earth. Understanding the origin and evolution of mechanistic innovations for...
Differential regulation of gene expression has produced the astonishing diversity of life on Earth. Understanding the origin and evolution of mechanistic innovations for control of gene expression is therefore integral to evolutionary and developmental biology. Cytoplasmic polyadenylation is the biochemical extension of polyadenosine at the 3'-end of cytoplasmic mRNAs. This process regulates the translation of specific maternal transcripts and is mediated by the Cytoplasmic Polyadenylation Element-Binding Protein family (CPEBs). Genes that code for CPEBs are amongst a very few that are present in animals but missing in nonanimal lineages. Whether cytoplasmic polyadenylation is present in non-bilaterian animals (i.e., sponges, ctenophores, placozoans, and cnidarians) remains unknown. We have conducted phylogenetic analyses of CPEBs, and our results show that CPEB1 and CPEB2 subfamilies originated in the animal stem lineage. Our assessment of expression in the sea anemone, Nematostella vectensis (Cnidaria), and the comb jelly, Mnemiopsis leidyi (Ctenophora), demonstrates that maternal expression of CPEB1 and the catalytic subunit of the cytoplasmic polyadenylation machinery (GLD2) is an ancient feature that is conserved across animals. Furthermore, our measurements of poly(A)-tail elongation reveal that key targets of cytoplasmic polyadenylation are shared between vertebrates, cnidarians, and ctenophores, indicating that this mechanism orchestrates a regulatory network that is conserved throughout animal evolution. We postulate that cytoplasmic polyadenylation through CPEBs was a fundamental innovation that contributed to animal evolution from unicellular life.
Topics: Animals; Phylogeny; Polyadenylation; Ctenophora; Sea Anemones
PubMed: 37288606
DOI: 10.1093/molbev/msad137 -
The New Phytologist Oct 2021Despite a much higher proportion of intragenic heterochromatin-containing genes in crop genomes, the importance of intragenic heterochromatin in crop development remains...
Despite a much higher proportion of intragenic heterochromatin-containing genes in crop genomes, the importance of intragenic heterochromatin in crop development remains unclear. Intragenic heterochromatin can be recognised by a protein complex, ASI1-AIPP1-EDM2 (AAE) complex, to regulate alternative polyadenylation. Here, we investigated the impact of rice ASI1 on global poly(A) site usage through poly(A) sequencing and ASI1-dependent regulation on rice development. We found that OsASI1 is essential for rice pollen development and flowering. OsASI1 dysfunction has an important impact on global poly(A) site usage, which is closely related to heterochromatin marks. Intriguingly, OsASI1 interacts with the intronic heterochromatin of OsXRNL, a nuclear XRN family exonuclease gene involved in the processing of an miRNA precursor, to promote the processing of full-length OsXRNL and regulate miRNA abundance. We found that OsASI1-mediated regulation of pollen development partially depends on OsXRNL. Finally, we characterised the rice AAE complex and its involvement in alternative polyadenylation and pollen development. Our findings help to elucidate an epigenetic mechanism governing miRNA abundance and rice development, and provide a valuable resource for studying the epigenetic mechanisms of many important processes in crops.
Topics: Gene Expression Regulation, Plant; Heterochromatin; MicroRNAs; Oryza; Pollen; Polyadenylation
PubMed: 34289124
DOI: 10.1111/nph.17635 -
Nucleic Acids Research Mar 2022Most human protein-coding genes produce alternative polyadenylation (APA) isoforms that differ in 3' UTR size or, when coupled with splicing, have variable coding...
Most human protein-coding genes produce alternative polyadenylation (APA) isoforms that differ in 3' UTR size or, when coupled with splicing, have variable coding sequences. APA is an important layer of gene expression program critical for defining cell identity. Here, by using a catalytically dead Cas9 and coupling its target site with polyadenylation site (PAS), we develop a method, named CRISPRpas, to alter APA isoform abundance. CRISPRpas functions by enhancing proximal PAS usage, whose efficiency is influenced by several factors, including targeting strand of DNA, distance between PAS and target sequence and strength of the PAS. For intronic polyadenylation (IPA), splicing features, such as strengths of 5' splice site and 3' splice site, also affect CRISPRpas efficiency. We show modulation of APA of multiple endogenous genes, including IPA of PCF11, a master regulator of APA and gene expression. In sum, CRISPRpas offers a programmable tool for APA regulation that impacts gene expression.
Topics: 3' Untranslated Regions; Gene Expression Regulation; Genetic Techniques; Humans; Introns; Polyadenylation; RNA Isoforms; RNA Splice Sites; RNA Splicing; RNA, Messenger; mRNA Cleavage and Polyadenylation Factors
PubMed: 34244761
DOI: 10.1093/nar/gkab519 -
Nature Communications Sep 2022Most genes in higher eukaryotes express isoforms with distinct 3' untranslated regions (3' UTRs), generated by alternative polyadenylation (APA). Since 3' UTRs are...
Most genes in higher eukaryotes express isoforms with distinct 3' untranslated regions (3' UTRs), generated by alternative polyadenylation (APA). Since 3' UTRs are predominant locations of post-transcriptional regulation, APA can render such programs conditional, and can also alter protein sequences via alternative last exon (ALE) isoforms. We previously used 3'-sequencing from diverse Drosophila samples to define multiple tissue-specific APA landscapes. Here, we exploit comprehensive single nucleus RNA-sequencing data (Fly Cell Atlas) to elucidate cell-type expression of 3' UTRs across >250 adult Drosophila cell types. We reveal the cellular bases of multiple tissue-specific APA/ALE programs, such as 3' UTR lengthening in differentiated neurons and 3' UTR shortening in spermatocytes and spermatids. We trace dynamic 3' UTR patterns across cell lineages, including in the male germline, and discover new APA patterns in the intestinal stem cell lineage. Finally, we correlate expression of RNA binding proteins (RBPs), miRNAs and global levels of cleavage and polyadenylation (CPA) factors in several cell types that exhibit characteristic APA landscapes, yielding candidate regulators of transcriptome complexity. These analyses provide a comprehensive foundation for future investigations of mechanisms and biological impacts of alternative 3' isoforms across the major cell types of this widely-studied model organism.
Topics: 3' Untranslated Regions; Animals; Drosophila; Male; Polyadenylation; Protein Isoforms; Sequence Analysis, RNA
PubMed: 36100597
DOI: 10.1038/s41467-022-32305-0