The 3'-end poly(A) tail is an important and potent feature of most mRNA molecules that affects mRNA fate and translation efficiency. Polyadenylation is a posttranscriptional process that occurs in the nucleus by canonical poly(A) polymerases (PAPs). In some specific instances, the poly(A) tail can also be extended in the cytoplasm by noncanonical poly(A) polymerases (ncPAPs). This epitranscriptomic regulation of mRNA recently became one of the most interesting aspects in the field. Advances in RNA sequencing technologies and software development have allowed the precise measurement of poly(A) tails, identification of new ncPAPs, expansion of the function of known enzymes, discovery and a better understanding of the physiological role of tail heterogeneity, and recognition of a correlation between tail length and RNA translatability. Here, we summarize the development of polyadenylation research methods, including classic low-throughput approaches, Illumina-based genome-wide analysis, and advanced state-of-art techniques that utilize long-read third-generation sequencing with Pacific Biosciences and Oxford Nanopore Technologies platforms. A boost in technical opportunities over recent decades has allowed a better understanding of the regulation of gene expression at the mRNA level. This article is categorized under: RNA Methods > RNA Analyses In Vitro and In Silico.

Siphophages have a long, flexible, and noncontractile tail that connects to the capsid through a neck. The phage tail is essential for host cell recognition and virus-host cell interactions; moreover, it serves as a channel for genome delivery during infection. However, the in situ high-resolution structure of the neck-tail complex of siphophages remains unknown. Here, we present the structure of the siphophage lambda "wild type," the most widely used, laboratory-adapted fiberless mutant. The neck-tail complex comprises a channel formed by stacked 12-fold and hexameric rings and a 3-fold symmetrical tip. The interactions among DNA and a total of 246 tail protein molecules forming the tail and neck have been characterized. Structural comparisons of the tail tips, the most diversified region across the lambda and other long-tailed phages or tail-like machines, suggest that their tail tip contains conserved domains, which facilitate tail assembly, receptor binding, cell adsorption, and DNA retaining/releasing. These domains are distributed in different tail tip proteins in different phages or tail-like machines. The side tail fibers are not required for the phage particle to orient itself vertically to the surface of the host cell during attachment.

Phage G is recognized as having a remarkably large genome and capsid size among isolated, propagated phages. Negative stain electron microscopy of the host-phage G interaction reveals tail sheaths that are contracted towards the distal tip and decoupled from the head-neck region. This is different from the typical myophage tail contraction, where the sheath contracts upward, while being linked to the head-neck region. Our cryo-EM structures of the non-contracted and contracted tail sheath show that: (1) The protein fold of the sheath protein is very similar to its counterpart in smaller, contractile phages such as T4 and phi812; (2) Phage G's sheath structure in the non-contracted and contracted states are similar to phage T4's sheath structure. Similarity to other myophages is confirmed by a comparison-based study of the tail sheath's helical symmetry, the sheath protein's evolutionary timetree, and the organization of genes involved in tail morphogenesis. Atypical phase G tail contraction could be due to a missing anchor point at the upper end of the tail sheath that allows the decoupling of the sheath from the head-neck region. Explaining the atypical tail contraction requires further investigation of the phage G sheath anchor points.

Tail docking of neonatal pigs is widely used as a measure to reduce the incidence of tail biting, a complex management problem in the pig industry. Concerns exist over the long-term consequences of tail docking for possible tail stump pain sensitivity due to the development of traumatic neuromas in injured peripheral nerves. Tail stumps were obtained post mortem from four female pigs at each of 1, 4, 8 and 16 weeks following tail amputation (approximately two-thirds removed) by a gas-heated docking iron on post natal day 3. Tissues were processed routinely for histopathological examination. Non-neural inflammatory and reparative epidermal and dermal changes associated with tissue thickening and healing were observed 1 to 4 months after docking. Mild neutrophilic inflammation was present in some cases, although this and other degenerative and non-neural reparative changes are not likely to have caused pain. Traumatic neuroma and neuromatous tissue development was not observed 1 week after tail docking, but was evident 1 month after tail docking. Over time there was marked nerve sheath and axonal proliferation leading to the formation of neuromata, which were either localized and circumscribed or comprised of multiple axons dispersed within granulation tissue. Four months after tail resection, neuroma formation was still incomplete, with possible implications for sensitivity of the tail stump.

Very little is known about the factors that cause variation in regenerative potential within and between species. Here, we used a genetic approach to identify heritable genetic factors that explain variation in tail regenerative outgrowth. A hybrid ambystomatid salamander (Ambystoma mexicanum x A. andersoni) was crossed to an A. mexicanum and 217 offspring were induced to undergo metamorphosis and attain terrestrial adult morphology using thyroid hormone. Following metamorphosis, each salamander's tail tip was amputated and allowed to regenerate, and then amputated a second time and allowed to regenerate. Also, DNA was isolated from all individuals and genotypes were determined for 187 molecular markers distributed throughout the genome. The area of tissue that regenerated after the first and second amputations was highly positively correlated across males and females. Males presented wider tails and regenerated more tail tissue during both episodes of regeneration. Approximately 66-68% of the variation in regenerative outgrowth was explained by tail width, while tail length and genetic sex did not explain a significant amount of variation. A small effect QTL was identified as having a sex-independent effect on tail regeneration, but this QTL was only identified for the first episode of regeneration. Several molecular markers significantly affected regenerative outgrowth during both episodes of regeneration, but the effect sizes were small (<4%) and correlated with tail width. The results show that ambysex and minor effect QTL explain variation in adult tail morphology and importantly, tail width. In turn, tail width at the amputation plane largely determines the rate of regenerative outgrowth. Because amputations in this study were made at approximately the same position of the tail, our results resolve an outstanding question in regenerative biology: regenerative outgrowth positively co-varies as a function of tail width at the amputation site.

The regeneration of the amputated tail of Xenopus laevis larvae is an excellent model system for regeneration research. The wound left by the amputated tail is covered with epidermis within 24 h. Then, the cell number increases near the amputation plane at the notochord, spinal cord and muscle regions. An apparently complete tail with notochord, muscle and spinal cord is regenerated within two weeks. To reveal whether the molecular mechanism underlying the tail regeneration is the same as that in embryonic tail development, the gene expression patterns of the embryonic tail bud and the regenerating tail were compared by in situ hybridization and reverse transcription-polymerase chain reaction. Most genes analyzed were expressed at similar levels in both tissues, whereas two bone morphogenetic protein (BMP)-antagonists, chordin and noggin, were detected only in the embryonic tail bud. The regenerating tail also lacked expression of Xshh in the floor plate and expression of Xdelta-1 in the spinal cord and presomitic mesoderm. These results show that there are some differences in gene expression between the two processes. Furthermore, when the tail of Xenopus larvae is amputated, the regenerating tail has a gene expression pattern similar to the distal portion of the larval tail rather than the embryonic tail bud, suggesting that the cut larval tail does not make a new embryonic tail bud, but rather a new larval tail tip for regeneration.

Desmosomal cadherins, desmogleins (Dsgs) and desmocollins, make up the adhesive core of intercellular junctions called desmosomes. A critical determinant of epithelial adhesive strength is the level and organization of desmosomal cadherins on the cell surface. The Dsg subclass of desmosomal cadherins contains a C-terminal unique region (Dsg unique region [DUR]) with unknown function. In this paper, we show that the DUR of Dsg2 stabilized Dsg2 at the cell surface by inhibiting its internalization and promoted strong intercellular adhesion. DUR also facilitated Dsg tail-tail interactions. Forced dimerization of a Dsg2 tail lacking the DUR led to decreased internalization, supporting the conclusion that these two functions of the DUR are mechanistically linked. We also show that a Dsg2 mutant, V977fsX1006, identified in arrhythmogenic right ventricular cardiomyopathy patients, led to a loss of Dsg2 tail self-association and underwent rapid endocytosis in cardiac muscle cells. Our observations illustrate a new mechanism desmosomal cadherins use to control their surface levels, a key factor in determining their adhesion and signaling roles.

This experiment assessed the efficacy of the cauterisation procedure with or without pain relief (injectable meloxicam) in mitigating the acute stress response to tail docking. Male piglets (n = 432) were allocated to the following treatments at 2-d post-farrowing: (1) no handling, (2) sham handling, (3) tail docked using clippers, (4) tail docked using a cauteriser, (5) meloxicam + clipper, and (6) meloxicam + cauteriser. Meloxicam treatments used Metacam® at 5 mg/mL injected i.m. 1 h prior to tail docking. Blood samples were collected at 15 and 30 min post-treatment and analysed for total plasma cortisol. Behaviours indicative of pain such as escape attempts, vocalisations and standing with head lowered were measured. The duration of vocalisations and frequency of escape attempts during treatment were greater in all tail docking treatments compared to the sham treatment. Piglets in the clipper treatment had higher (p < 0.05) cortisol concentrations at 30 min but not 15 min after treatment and stood for longer (p < 0.001) with head lowered in the first 60 min after treatment than those in the cauterisation treatment. Meloxicam reduced (p < 0.05) both the cortisol response at 30 min after tail docking with the clipper as well as the behavioural response in the first 60 min after tail docking with the clipper. In comparison to the sham treatment, cortisol concentrations at 15 min were higher in the two tail docking treatments whereas the tail docking treatments with meloxicam were similar to the sham handling treatment. In comparison to the sham handling treatment, cortisol concentrations at 30 min post-docking were higher (p < 0.05) only in the clipper treatment. While cauterisation appears to be less aversive than the clipper procedure, the administration of meloxicam did not mitigate the behavioural response during tail docking using either procedure, but reduced standing with head lowered in the first hour after docking for both methods. The commercial viability of administration of meloxicam requires consideration before it is recommended for use compared to cauterisation alone, as it requires additional handling of piglets and costs.

This experiment assessed the efficacy of the cauterisation procedure with or without pain relief (injectable meloxicam) in mitigating the acute stress response to tail docking. Male piglets (n = 432) were allocated to the following treatments at 2-d post-farrowing: (1) no handling, (2) sham handling, (3) tail docked using clippers, (4) tail docked using a cauteriser, (5) meloxicam + clipper, and (6) meloxicam + cauteriser. Meloxicam treatments used Metacam® at 5 mg/mL injected i.m. 1 h prior to tail docking. Blood samples were collected at 15 and 30 min post-treatment and analysed for total plasma cortisol. Behaviours indicative of pain such as escape attempts, vocalisations and standing with head lowered were measured. The duration of vocalisations and frequency of escape attempts during treatment were greater in all tail docking treatments compared to the sham treatment. Piglets in the clipper treatment had higher (p < 0.05) cortisol concentrations at 30 min but not 15 min after treatment and stood for longer (p < 0.001) with head lowered in the first 60 min after treatment than those in the cauterisation treatment. Meloxicam reduced (p < 0.05) both the cortisol response at 30 min after tail docking with the clipper as well as the behavioural response in the first 60 min after tail docking with the clipper. In comparison to the sham treatment, cortisol concentrations at 15 min were higher in the two tail docking treatments whereas the tail docking treatments with meloxicam were similar to the sham handling treatment. In comparison to the sham handling treatment, cortisol concentrations at 30 min post-docking were higher (p < 0.05) only in the clipper treatment. While cauterisation appears to be less aversive than the clipper procedure, the administration of meloxicam did not mitigate the behavioural response during tail docking using either procedure, but reduced standing with head lowered in the first hour after docking for both methods. The commercial viability of administration of meloxicam requires consideration before it is recommended for use compared to cauterisation alone, as it requires additional handling of piglets and costs.

SINEs, non-autonomous short retrotransposons, are widespread in mammalian genomes. Their transcripts are generated by RNA polymerase III (pol III). Transcripts of certain SINEs can be polyadenylated, which requires polyadenylation and pol III termination signals in their sequences. Our sequence analysis divided Can SINEs in canids into four subfamilies, older a1 and a2 and younger b1 and b2. Can_b2 and to a lesser extent Can_b1 remained retrotranspositionally active, while the amplification of Can_a1 and Can_a2 ceased long ago. An extraordinarily high Can amplification was revealed in different dog breeds. Functional polyadenylation signals were analyzed in Can subfamilies, particularly in fractions of recently amplified, i.e., active copies. The transcription of various Can constructs transfected into HeLa cells proposed AATAAA and (TC)n as functional polyadenylation signals. Our analysis indicates that older Can subfamilies (a1, a2, and b1) with an active transcription terminator were amplified by the T+ mechanism (with polyadenylation of pol III transcripts). In the currently active Can_b2 subfamily, the amplification mechanisms with (T+) and without the polyadenylation of pol III transcripts (T-) irregularly alternate. The active transcription terminator tends to shorten, which renders it nonfunctional and favors a switch to the T- retrotransposition. The activity of a truncated terminator is occasionally restored by its elongation, which rehabilitates the T+ retrotransposition for a particular SINE copy.

Tails are an intricate component of the locomotor system for many vertebrates. Leopard geckos (Eublepharis macularius) possess a large tail that is laterally undulated during steady locomotion. However, the tail is readily shed via autotomy, resulting in the loss of tail function, loss in body mass, and a cranial shift in the center of mass. To elucidate the function of tail undulations, we investigated changes in limb kinematics after manipulating the tail artificially by restricting tail undulations and naturally by removing the tail via autotomy. Restricting tail undulations resulted in kinematic adjustments similar to those that occur following tail autotomy, characterized by more flexed hind limb joints. These data suggest that effects of autotomy on locomotion may be linked to the loss of tail movements rather than the loss of mass or a shift in center of mass. We also provide empirical support for the link between lateral tail undulations and step length through the rotation of the pelvic girdle and retraction of the femur. Restriction and autotomy of the tail limits pelvic rotation, which reduces femur retraction and decreases step length. Our findings demonstrate a functional role for tail undulations in geckos, which likely applies to other terrestrial vertebrates.

The T5 family of viruses are tailed bacteriophages characterized by a long non-contractile tail. The bacteriophage DT57C is closely related to the paradigmal T5 phage, though it recognizes a different receptor (BtuB) and features highly divergent lateral tail fibers (LTF). Considerable portions of T5-like phages remain structurally uncharacterized. Here, we present the structure of DT57C determined by cryo-EM, and an atomic model of the virus, which was further explored using all-atom molecular dynamics simulations. The structure revealed a unique way of LTF attachment assisted by a dodecameric collar protein LtfC, and an unusual composition of the phage neck constructed of three protein rings. The tape measure protein (TMP) is organized within the tail tube in a three-stranded parallel α-helical coiled coil which makes direct contact with the genomic DNA. The presence of the C-terminal fragment of the TMP that remains within the tail tip suggests that the tail tip complex returns to its original state after DNA ejection. Our results provide a complete atomic structure of a T5-like phage, provide insights into the process of DNA ejection as well as a structural basis for the design of engineered phages and future mechanistic studies.

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a plasma membrane protein ubiquitously present in humans. It regulates intracellular pH by removing an intracellular proton in exchange for an extracellular sodium. It consists of a 500 amino acid membrane domain plus a 315 amino acid, regulatory cytosolic tail. Here, we investigated the effect of mutation of two amino acids of the regulatory tail, Ser785 and Ser787, that were similar in location and context to two amino acids of the Arabidopsis Na+/H+ exchanger SOS1. Mutation of these two amino acids to either Ala or phosphomimetic Glu did not affect surface targeting but led to a slight reduction in the level of protein expressed. The activity of the NHE1 protein was reduced in the phosphomimetic mutations and the effect was due to a decrease in Vmax activity. The Ser to Glu mutations also caused a change in the apparent molecular weight of both the full-length protein and of the cytosolic tail of NHE1. A conformational change in this region was indicated by differential trypsin sensitivity. We also found that a peptide containing amino acids 783-790 bound to several more proximal regions of the NHE1 tail in in vitro protein interaction experiments. The results are the first characterization of these two amino acids and show that they have significant effects on enzyme kinetics and the structure of the NHE1 protein.

Bacteriophage lambda has a double-stranded DNA genome and a long, flexible, non-contractile tail encoded by a contiguous block of 11 genes downstream of the head genes. The tail allows host recognition and delivery of viral DNA from the head shell to the cytoplasm of the infected cell. Here, we present a high-resolution structure of the tail complex of bacteriophage lambda determined by cryoelectron microscopy. Most component proteins of the lambda tail were determined at the atomic scale. The structure sheds light on the molecular organization of the extensively studied tail of bacteriophage lambda.

Tail biting is a major welfare and economic problem for indoor pig producers worldwide. Low tail posture is an early warning sign which could reduce tail biting unpredictability. Taking a precision livestock farming approach, we used Time-of-flight 3D cameras, processing data with machine vision algorithms, to automate the measurement of pig tail posture. Validation of the 3D algorithm found an accuracy of 73.9% at detecting low vs. not low tails (Sensitivity 88.4%, Specificity 66.8%). Twenty-three groups of 29 pigs per group were reared with intact (not docked) tails under typical commercial conditions over 8 batches. 15 groups had tail biting outbreaks, following which enrichment was added to pens and biters and/or victims were removed and treated. 3D data from outbreak groups showed the proportion of low tail detections increased pre-outbreak and declined post-outbreak. Pre-outbreak, the increase in low tails occurred at an increasing rate over time, and the proportion of low tails was higher one week pre-outbreak (-1) than 2 weeks pre-outbreak (-2). Within each batch, an outbreak and a non-outbreak control group were identified. Outbreak groups had more 3D low tail detections in weeks -1, +1 and +2 than their matched controls. Comparing 3D tail posture and tail injury scoring data, a greater proportion of low tails was associated with more injured pigs. Low tails might indicate more than just tail biting as tail posture varied between groups and over time and the proportion of low tails increased when pigs were moved to a new pen. Our findings demonstrate the potential for a 3D machine vision system to automate tail posture detection and provide early warning of tail biting on farm.

Thermus thermophilus bacteriophage P23-45 encodes a giant 5,002-residue tail tape measure protein (TMP) that defines the length of its extraordinarily long tail. Here, we show that the N-terminal portion of P23-45 TMP is an unusual RNA polymerase (RNAP) homologous to cellular RNAPs. The TMP-fused virion RNAP transcribes pre-early phage genes, including a gene that encodes another, non-virion RNAP, that transcribes early and some middle phage genes. We report the crystal structures of both P23-45 RNAPs. The non-virion RNAP has a crab-claw-like architecture. By contrast, the virion RNAP adopts a unique flat structure without a clamp. Structure and sequence comparisons of the P23-45 RNAPs with other RNAPs suggest that, despite the extensive functional differences, the two P23-45 RNAPs originate from an ancient gene duplication in an ancestral phage. Our findings demonstrate striking adaptability of RNAPs that can be attained within a single virus species.

The chromodomain of HP1α binds directly to lysine 9-methylated histone H3 (H3K9me). This interaction is enhanced by phosphorylation of serine residues in the N-terminal tail of HP1α by unknown mechanism. Here we show that phosphorylation modulates flexibility of HP1α's N-terminal tail, which strengthens the interaction with H3. NMR analysis of HP1α's chromodomain with N-terminal tail reveals that phosphorylation does not change the overall tertiary structure, but apparently reduces the tail dynamics. Small angle X-ray scattering confirms that phosphorylation contributes to extending HP1α's N-terminal tail. Systematic analysis using deletion mutants and replica exchange molecular dynamics simulations indicate that the phosphorylated serines and following acidic segment behave like an extended string and dynamically bind to H3 basic residues; without phosphorylation, the most N-terminal basic segment of HP1α inhibits interaction of the acidic segment with H3. Thus, the dynamic string-like behavior of HP1α's N-terminal tail underlies the enhancement in H3 binding due to phosphorylation.

The tail of the Xenopus tadpole will regenerate following amputation, and all three of the main axial structures - the spinal cord, the notochord and the segmented myotomes - are found in the regenerated tail. We have investigated the cellular origin of each of these three tissue types during regeneration. We produced Xenopus laevis embryos transgenic for the CMV (Simian Cytomegalovirus) promoter driving GFP (Green Fluorescent Protein) ubiquitously throughout the embryo. Single tissues were then specifically labelled by making grafts at the neurula stage from transgenic donors to unlabelled hosts. When the hosts have developed to tadpoles, they carry a region of the appropriate tissue labelled with GFP. These tails were amputated through the labelled region and the distribution of labelled cells in the regenerate was followed. We also labelled myofibres using the Cre-lox method. The results show that the spinal cord and the notochord regenerate from the same tissue type in the stump, with no labelling of other tissues. In the case of the muscle, we show that the myofibres of the regenerate arise from satellite cells and not from the pre-existing myofibres. This shows that metaplasia between differentiated cell types does not occur, and that the process of Xenopus tail regeneration is more akin to tissue renewal in mammals than to urodele tail regeneration.

MR1 is a conserved molecule that binds microbial vitamin B metabolites and presents them to unconventional T cells. Lim and colleagues (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202110125) uncover the role of AP2 in ensuring MR1 surface presentation, which relies on an atypical motif within the MR1 cytoplasmic tail.

Abnormal tail biting behaviour is a major welfare problem for pigs receiving the behaviour, as well as an indication of decreased welfare in the pigs performing it. However, not all pigs in a pen perform or receive tail biting behaviour and it has recently been shown that these 'neutral' pigs not only differ in their behaviour, but also in their gene expression compared to performers and receivers of tail biting in the same pen. To investigate whether this difference was linked to the cause or a consequence of them not being involved in the outbreak of tail biting, behaviour and brain gene expression was compared with 'control' pigs housed in pens with no tail biting. It was shown that the pigs housed in control pens performed a wider variety of pig-directed abnormal behaviour (belly nosing 0.95±1.59, tail in mouth 0.31±0.60 and 'other' abnormal 1.53±4.26; mean±S.D) compared to the neutral pigs (belly nosing 0.30±0.62, tail in mouth 0.13±0.50 and "other" abnormal 0.42±1.06). With Affymetrix gene expression arrays, 107 transcripts were identified as differently expressed (p<0.05) between these two categories of pigs. Several of these transcripts had already been shown to be differently expressed in the neutral pigs when they were compared to performers and receivers of tail biting in the same pen in an earlier study. Hence, the different expression of these genes cannot be a consequence of the neutral pigs not being involved in tail biting behaviour, but rather linked to the cause contributing to why they were not involved in tail biting interactions. These neutral pigs seem to have a genetic and behavioural profile that somehow contributes to them being resistant to performing or receiving pig-directed abnormal behaviour, such as tail biting, even when housed in an environment that elicits that behaviour in other pigs.