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Results for “Poly(A) tail dynamics”

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PubMed2025

Dynamic Evolution of Poly-A Tail Lengths Visualized by RNAse H Assay and Northern Blot Using Nonradioactive Probes in Yeast.

Poly-A tail length dynamics have been extensively studied from yeast to human, mostly using reporter transcripts. Recent studies have been carried out genome-wide to determine the status of poly-A tails at steady state. However, poly-A tail measurement at equilibrium gives an overall length that reflects a mixture of the different poly-A tail sizes for a single transcript. New genome-scale techniques are emerging to estimate dynamic of poly-A tails lengths, but they are not yet routine and individual validation experiments are useful. In this chapter we describe a protocol for visualizing poly-A tail lengths following transcription inhibition for a reporter mRNA using denaturing poly-acrylamide gel electrophoresis and northern blot assay. This protocol is quick to set up, requires the purchase of only a few specific reagents, does not rely on radioactivity for RNA monitoring, and can be easily implemented in any molecular biology laboratory.

Poly A↗
PubMed2026

Maternal PAN2 selectively maintains mRNA Poly(A) tail homeostasis to regulate RNA degradation during oocyte-to-early embryo transition in mice.

In mammals, the precise degradation of maternal mRNAs is essential for oocyte maturation and early embryonic development, as it facilitates the "maternal-to-zygotic transition (MZT)" by eliminating maternal transcripts and enabling zygotic genome activation (ZGA). However, the physiological role of the poly(A)-specific nuclease 2 (PAN2), a deadenylase that initiates cascade degradation of long-tailed transcripts, remains unknown. Here, we generated oocyte-specific Pan2 conditional knockout (cKO) mice to investigate its role. We found that Pan2 cKO females exhibit severe female subfertility despite normal oocyte maturation and ovulation, with embryos arresting at the 2-cell stage. PAIso-seq2 and transcriptome sequencing reveal that PAN2 coordinates maternal mRNA deadenylation and decay. Mechanistically, PAN2 recognizes its substrates through a PAN3-PABPC1 bridging complex, and it preferentially targets transcripts whose poly(A) tails lack guanosine (G) but are enriched for uridine (U). PAN2 deficiency causes poly(A) tail dyshomeostasis, leading to global accumulation of maternal mRNAs, impaired zygotic genome activation, and abnormal protein accumulation in 2-cell embryos. Overexpression of these proteins phenocopies developmental defects. Notably, the PAN2-regulated transcriptome is largely non-overlapping with the LC3B-mediated degradation pathway, highlighting the unique and non-redundant role of PAN2 in maternal mRNA clearance. Our study establishes maternal PAN2 as a critical regulator of poly(A) tail homeostasis, ensuring timely maternal mRNA clearance and proper ZGA, highlighting the stage-specific and tail-composition-dependent functions of the deadenylase cascade during the maternal-to-zygotic transition. These findings offer new perspectives on post-transcriptional regulatory mechanisms in early mammalian embryogenesis.

Deadenylation↗
PubMed1977

Cytoplasmic nonpolysomal ribonucleoprotein particles in sea urchin embryos and their relationship to protein synthesis.

We have examined the relationship between the newly synthesized mRNA that enters polysomes in sea urchin embryos and the messengerlike RNA that enters the pool of ribosome-free ribonucleoprotein particles (free RNPs or informosomes). Although the RNA in the free RNPs turns over 25% more rapidly than in the polysomes, labeling kinetics indicate that the RNA containing poly(A) [poly(A)(+)RNA] and the RNA not containing poly(A) [poly(A)(-)RNA] within each cytoplasmic compartment have very similar half-lives. The poly(A)(+)RNA from both free RNPs and polysomes binds ribosomes almost equally well in a reticulocyte lysate, and this binding is sensitive to inhibitors of initiation. The poly(A)(-)RNA from polysomes initiates as well as poly(A)(+)RNA; however, poly(A)(-)RNA from free RNPs is only half as efficient in binding to ribosomes, and by this criterion is only 50% mRNA. We have also examined the size and dynamics of shortening of the poly(A) tails of poly(A)(+)RNA from free RNPs and polysomes. Pulse-labeled poly(A) from both free RNPs and polysomes is about 180 nucleotides in length. Poly(A) shortening is very rapid in polysomes; steady-state labeled polysomal RNA is largely devoid of the 180-nucleotide-long poly(A) segments. Poly(A) shortening in free RNPs is slower; half of the poly(A) derived from steady-state free RNPs is still 180 nucleotides long. Despite this difference in the rates of poly(A) shortening, polysomes and free RNPs have very similar half-lives. There is, then, no obvious relationship between poly(A) shortening and turnover of mRNA in these embryos. The data are interpreted to mean that poly(A)(+)RNA from free RNPs is enriched for a class of mRNA that initiates less frequently in vivo than the bulk of the cellular mRNA.

Animals↗
PubMed2026

Uncovering hidden complexity in the Apis mellifera mitotranscriptome: a polyadenylation-centered perspective.

Mitochondrial transcription is gaining increasing attention as researchers seek to better understand the full coding potential of mitochondrial DNA (mtDNA). Emerging evidence suggests that mtDNA may encode additional elements beyond classical oxidative phosphorylation genes, pointing to a more complex transcriptional architecture than previously recognized. In this study, we explored the mitochondrial transcriptome of Apis mellifera (Insecta: Hymenoptera), with a particular focus on polyadenylation-associated features. Our analysis revealed that both sense and antisense transcripts undergo polyadenylation, although transcript abundance and poly(A) tail lengths varied markedly across mitochondrial genes. Several transcripts exhibited alternative isoforms, either extended or truncated, frequently including intergenic regions. These regions may represent functional non-coding elements or structural variants rather than conventional untranslated regions (UTRs). Interestingly, some transcripts also contained non-templated nucleotide additions particularly cytosine residues immediately upstream of the poly(A) tails. Monocistronic units that included portions of downstream intergenic regions were among the most abundantly represented, suggesting a possible regulatory role for these sequences. To experimentally validate our in silico findings, we performed RT-qPCR to assess relative gene expression and applied 3' RACE-PCR to define transcript boundaries. These approaches confirmed the presence of multiple transcript isoforms and supported the involvement of polyadenylation in shaping mitochondrial RNA diversity. Together, our findings reveal a previously underappreciated level of complexity in the A. mellifera mitochondrial transcriptome and highlight the potential regulatory significance of polyadenylation dynamics and intergenic region transcription.

Animals↗