Poly(A) tails are important elements in mRNA translation and stability. However, recent genome-wide studies concluded that poly(A) tail length was generally not associated with translational efficiency in non-embryonic cells. To investigate if poly(A) tail size might be coupled to gene expression in an intact organism, we used an adapted TAIL-seq protocol to measure poly(A) tails in Caenorhabditis elegans. Surprisingly, we found that well-expressed transcripts contain relatively short, well-defined tails. This attribute appears dependent on translational efficiency, as transcripts enriched for optimal codons and ribosome association had the shortest tail sizes, while non-coding RNAs retained long tails. Across eukaryotes, short tails were a feature of abundant and well-translated mRNAs. Although this seems to contradict the dogma that deadenylation induces translational inhibition and mRNA decay, it instead suggests that well-expressed mRNAs accumulate with pruned tails that accommodate a minimal number of poly(A) binding proteins, which may be ideal for protective and translational functions.
Müller glial cells are the source of retinal regeneration in fish and birds; although this process is efficient in fish, it is less so in birds and very limited in mammals. It has been proposed that factors necessary for providing neurogenic competence to Müller glia in fish and birds after retinal injury are not expressed in mammals. One such factor, the proneural transcription factor Ascl1, is necessary for retinal regeneration in fish but is not expressed after retinal damage in mice. We previously reported that forced expression of Ascl1 in vitro reprograms Müller glia to a neurogenic state. We now test whether forced expression of Ascl1 in mouse Müller glia in vivo stimulates their capacity for retinal regeneration. We find that transgenic expression of Ascl1 in adult Müller glia in undamaged retina does not overtly affect their phenotype; however, when the retina is damaged, the Ascl1-expressing glia initiate a response that resembles the early stages of retinal regeneration in zebrafish. The reaction to injury is even more pronounced in Müller glia in young mice, where the Ascl1-expressing Müller glia give rise to amacrine and bipolar cells and photoreceptors. DNaseI-seq analysis of the retina and Müller glia shows progressive reduction in accessibility of progenitor gene cis-regulatory regions consistent with the reduction in their reprogramming. These results show that at least one of the differences between mammal and fish Müller glia that bears on their difference in regenerative potential is the proneural transcription factor Ascl1.reprogramming | glia | regeneration | neurogenesis | eye
MicroRNAs (miRNAs) are small RNAs that guide Argonaute (AGO) proteins to specific target messenger RNAs (mRNAs) to repress their translation and stability. Canonically, miRNA targeting is reliant on base pairing of the seed region, nucleotides (nts) 2-7, of the miRNA to sites in mRNA 3'UTRs. Recently, the 3' half of the miRNA has gained attention for newly appreciated roles in regulating target specificity and regulation. Additionally, the extent of pairing to the miRNA 3'end can influence the stability of the miRNA itself. These findings highlight the importance of sequences beyond the seed in controlling the function and existence of miRNAs.
Argonaute (AGO) proteins partner with microRNAs (miRNAs) to target specific genes for post-transcriptional regulation. During larval development in Caenorhabditis elegans, Argonaute-Like Gene 1 (ALG-1) is the primary mediator of the miRNA pathway, while the related ALG-2 protein is largely dispensable. Here we show that in adult C. elegans these AGOs are differentially expressed and, surprisingly, work in opposition to each other; alg-1 promotes longevity, whereas alg-2 restricts lifespan. Transcriptional profiling of adult animals revealed that distinct miRNAs and largely non-overlapping sets of protein-coding genes are misregulated in alg-1 and alg-2 mutants. Interestingly, many of the differentially expressed genes are downstream targets of the Insulin/ IGF-1 Signaling (IIS) pathway, which controls lifespan by regulating the activity of the DAF-16/ FOXO transcription factor. Consistent with this observation, we show that daf-16 is required for the extended lifespan of alg-2 mutants. Furthermore, the long lifespan of daf-2 insulin receptor mutants, which depends on daf-16, is strongly reduced in animals lacking alg-1 activity. This work establishes an important role for AGO-mediated gene regulation in aging C. elegans and illustrates that the activity of homologous genes can switch from complementary to antagonistic, depending on the life stage.
Diseases and damage to the retina lead to losses in retinal neurons and eventual visual impairment. Although the mammalian retina has no inherent regenerative capabilities, fish have robust regeneration from Müller glia (MG). Recently, we have shown that driving expression of Ascl1 in adult mouse MG stimulates neural regeneration. the regeneration observed in the mouse is limited in the variety of neurons that can be derived from MG; Ascl1-expressing MG primarily generate bipolar cells. To better understand the limits of MG-based regeneration in mouse retinas, we used ATAC-and RNA-seq to compare newborn progenitors, immature MG (P8-P12), and mature MG. Our analysis demonstrated developmental differences in gene expression and accessible chromatin between progenitors and MG, primarily in neurogenic genes. overexpression of Ascl1 is more effective in reprogramming immature MG, than mature MG, consistent with a more progenitor-like epigenetic landscape in the former. We also used ASCL1 ChIPseq to compare the differences in ASCL1 binding in progenitors and reprogrammed MG. We find that bipolar-specific accessible regions are more frequently linked to bHLH motifs and ASCL1 binding. Overall, our analysis indicates a loss of neurogenic gene expression and motif accessibility during glial maturation that may prevent efficient reprogramming. The death of retinal neurons leads to permanent vision loss. While some species are readily capable of regenerating lost neurons, mammalian retinas are not. In mammals, neuron loss leads to reactive gliosis of the Müller glia (MG), similar to that of astrocytes in the brain 1. Teleost fish, by contrast, are capable of regenerating retinal neurons, including photoreceptors and ganglion cells, after damage. This regeneration is carried out by the MG, which respond to damage by generating progenitor-like cells, similar to those in the developing retina 2,3. Regeneration is accompanied by changes in gene expression and morphological changes to the MG, potentially regulated by epigenomic changes. The murine retina also undergoes epigenomic changes after damage, but neurogenic programs are not re-expressed, and neuronal regeneration does not occur 4. A critical difference between fish and mammalian MG in their response to damage is in their expression of the proneural transcription factor Ascl1. In fish, Ascl1 is quickly upregulated after damage, and is necessary for regeneration of new neurons 5,6. In the murine retina, Ascl1 is expressed in retinal progenitors and necessary for development of rods and bipolar cells 7 ; however it is not expressed in mature MG; moreover, after damage or in disease models, mouse MG do not spontaneously upregulate Ascl1 1,8. We recently directed Ascl1 expression to mouse MG with a inducible transgenic approach to test whether Ascl1 expression is sufficient to induce regeneration. Expression of Ascl1 in the MG of young mice (12 days post-natal (P12)) stimulated MG to generate new
The primary glial cells in the retina, the Müller glia, differentiate from retinal progenitors in the first postnatal week. CNTF/LIF/STAT3 signaling has been shown to promote their differentiation; however, another key glial differentiation signal, BMP, has not been examined during this period of Müller glial differentiation. In the course of our analysis of the BMP signaling pathway, we observed a transient wave of Smad1/5/8 signaling in the inner nuclear layer at the end of the first postnatal week, from postnatal day (P) 5 to P9, after the end of neurogenesis. To determine the function of this transient wave, we blocked BMP signaling during this period in vitro or in vivo, using either a BMP receptor antagonist or noggin (Nog). Either treatment leads to a reduction in expression of the Müller glia-specific genes Rlbp1 and Glul, and the failure of many of the Müller glia to repress the bipolar/photoreceptor gene Otx2. These changes in normal Müller glial differentiation result in permanent disruption of the retina, including defects in the outer limiting membrane, rosette formation and a reduction in functional acuity. Our results thus show that Müller glia require a transient BMP signal at the end of neurogenesis to fully repress the neural gene expression program and to promote glial gene expression.
19Diseases and damage to the retina lead to losses in retinal neurons and eventual visual 20 impairment. Although the mammalian retina has no inherent regenerative capabilities, fish have 21 robust regeneration from Müller glia (MG). Recently, we have shown that driving expression of 22 Ascl1 in adult mouse MG stimulates neurogenesis similar to fish regeneration. The regeneration 23 observed in the mouse is limited in the variety of neurons that can be derived from MG; Ascl1-24 expressing MG primarily generate bipolar cells. To better understand the limits of MG-based 25 regeneration in mouse retinas, we used ATAC-and RNA-seq to compare newborn progenitors 26 with MG. Our analysis demonstrated striking similarities between MG and progenitors, with 27 losses in regulatory motifs for neurogenesis genes. Young MG were found to have intermediate 28 expression profiles and accessible DNA, which is mirrored in the ability of Ascl1 to direct 29 bipolar neurogenesis in young MG. When comparing what makes bipolar and photoreceptor 30 cells distinct from glial cells, we find that bipolar-specific accessible regions are more frequently 31 linked to bHLH motifs and Ascl1 binding, indicating that Ascl1 preferentially binds to bipolar 32 regions. Overall, our analysis indicates a loss of neurogenic gene expression and motif 33 accessibility during glial maturation that may prevent efficient reprogramming. 34 35 36 Neuron loss caused by disease and damage to the mammalian retina can lead to 37 permanent vision loss. While some species are readily capable of regenerating lost neurons, 38 mammalian retinas are not regenerative. In the mammalian retina, neuron loss caused by direct 39 damage to the retina leads to reactive gliosis of the Müller glia (MG), similar to that of astrocytes 40 in the brain (Bringmann et al. 2009).41 Teleost fish, by contrast, are capable of regenerating retinal neurons, including 42 photoreceptors and ganglion cells, after damage. This regeneration is carried out by the MG, 43 which respond to damage by generating progenitor-like cells, similar to those in the developing 44 retina (Goldman 2014; Gemberling et al. 2013). This regeneration is accompanied by waves of 45 gene expression and morphological changes to the MG, regulated by epigenomic changes 46 directing regeneration (for review see VandenBosch and Reh 2019). The murine retina also 47 undergoes epigenomic changes after damage, but neurogenic programs are not re-expressed, and 48 neuronal regeneration does not occur (VandenBosch and Reh 2019). 49A critical difference between the fish MG and the mammalian MG in their response to 50 damage is in their expression of the proneural transcription factor Ascl1. In fish, Ascl1 is quickly 51 upregulated after damage, and is necessary for regeneration of new neurons (Ramachandran et 52 al. 2010; Fausett et al. 2008). In the murine retina, Ascl1 is expressed in retinal progenitors and 53 necessary for development of rods and bipolar cells (Ohsawa and Kageyama 2008); however it is 54 not expressed in...
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