Post-transcriptional cleavage and polyadenylation of messenger and long noncoding RNAs is coordinated by a supercomplex of ~20 individual proteins within the eukaryotic nucleus1,2. Polyadenylation plays an essential role in controlling RNA transcript stability, nuclear export, and translation efficiency3–6. More than half of all human RNA transcripts contain multiple polyadenylation signal sequences that can undergo alternative cleavage and polyadenylation during development and cellular differentiation7,8. Alternative cleavage and polyadenylation is an important mechanism for the control of gene expression and defects in 3’ end processing can give rise to myriad human diseases9,10. Here we show that fusion of catalytically dead Cas13 to a single mammalian polyadenylation factor, Nudix hydrolase 21 (NUDT21), allows for site-specific CRISPR-Cas13-guided cleavage and polyadenylation of RNA in mammalian cells. This approach, which we named Postscriptr, can be utilized for the non-genomic manipulation of gene expression and may have potential future therapeutic applications for treating human RNA processing diseases.

Skeletal muscle possesses a robust capacity to regenerate functional architectures with a unidirectional orientation. In this study, we successfully arranged skeletal myoblast (C2C12) cells along micropatterned gold strips on which chitohexaose was deposited via a vectorial chain immobilization approach. Hexa-N-acetyl-d-glucosamine (GlcNAc6) was site-selectively modified at its reducing end with thiosemicarbazide, then immobilized on a gold substrate in striped micropatterns via S–Au chemisorption. Gold micropatterns ranged from 100 to 1000 µm in width. Effects of patterning geometries on C2C12 cell alignment, morphology, and gene expression were investigated. Unidirectional alignment of C2C12 cells having GlcNAc6 receptors was clearly observed along the micropatterns. Decreasing striped pattern width increased cell attachment and proliferation, suggesting that the fixed GlcNAc6 and micropatterns impacted cell function. Possibly, interactions between nonreducing end groups of fixed GlcNAc6 and cell surface receptors initiated cellular alignment. Our technique for mimicking native tissue organization should advance applications in tissue engineering.

Myoblast fusion into functionally-distinct myotubes to form in vitro skeletal muscle constructs under differentiation serum-free conditions still remains a challenge. Herein, we report that our microtopographical carbohydrate substrates composed of bioactive hexa-N-acetyl-d-glucosamine (GlcNAc6) modulated the efficiency of myoblast fusion without requiring horse serum or any differentiation medium during cell culture. Promotion of the differentiation of dissociated mononucleated skeletal myoblasts (C2C12; a mouse myoblast cell line) into robust myotubes was found only on GlcNAc6 micropatterns, whereas the myoblasts on control, non-patterned GlcNAc6 substrates or GlcNAc6-free patterns exhibited an undifferentiated form. We also examined the possible role of GlcNAc6 micropatterns with various widths in the behavior of C2C12 cells in early and late stages of myogenesis through mRNA expression of myosin heavy chain (MyHC) isoforms. The spontaneous contraction of myotubes was investigated via the regulation of glucose transporter type 4 (GLUT4), which is involved in stimulating glucose uptake during cellular contraction. Narrow patterns demonstrated enhanced glucose uptake rate and generated a fast-twitch muscle fiber type, whereas the slow-twitch muscle fiber type was dominant on wider patterns. Our findings indicated that GlcNAc6-mediated integrin interactions are responsible for guiding myoblast fusion forward along with myotube formation.

Human monogenetic diseases can arise from the aberrant expansion of tandem nucleotide repeat sequences, which when transcribed into RNA, can misfold and aggregate into toxic nuclear foci 1 . Nuclear retention of repeat-containing RNAs can disrupt their normal expression and induce widespread splicing defects by sequestering essential RNA binding proteins. Among the most prevalent of these disorders is myotonic dystrophy type 1 (DM1), a disease occurring from the expression of a noncoding CTG repeat expansion in the 3'UTR of the human dystrophia myotonica protein kinase (DMPK) gene 2,3 . Here we show that RNAbinding CRISPR-Cas13, with a robust non-classical nuclear localization signal, can be efficiently targeted to toxic nuclear RNA foci for either visualization or cleavage, tools we named hilightR and eraseR, respectively. HilightR combines catalytically dead Cas13b (dCas13b) with a fluorescent protein to directly visualize CUG repeat RNA foci in the nucleus of live cells, allowing for quantification of foci number and observation of foci dynamics. EraseR utilizes the intrinsic endoribonuclease activity of Cas13b, targeted to nuclear CUG repeat RNA, to disrupt nuclear foci. These studies demonstrate the potential for targeting toxic nuclear RNA foci directly with CRISPR-Cas13 for either the identification or treatment of DM1. The efficient and sequence programmable nature of CRISPR-Cas13 systems will allow for rapid targeting and manipulation of other human nuclear RNA disorders, without the associated risks of genome editing.Myotonic dystrophy type 1 (DM1) is an autosomal dominant human monogenic disease characterized by progressive myotonia, muscle wasting, cardiac arrhythmias, and cognitive dysfunction 4 . DM1 is the most common form of adult-onset muscular

Endogenous retroelements (REs) are pervasive in mammalian genomes, and consist mostly of silenced mobile genetic sequences long considered to be ‘junk DNA.’ In a few known instances, mammalian genomes have captured and repurposed endogenous retroviral sequences to play important roles in regulating mammalian cell functions, notably cell fusion. Our previous work has shown that some mammalian transcripts misannotated as lncRNAs can be translated into small functional peptides, called micropeptides. Here we report the discovery of a cardiac-specific micropeptide encoded by a misannotated lncRNA transcribed from a murine endogenous retroviral element. We named this transcript CARDI, and the encoded micropeptide CARDI-B, since translation of the micropeptide occurs from the second ATG start codon. We show that CARDI-B shares homology with a singular domain of a retroviral protein and localizes to the nucleus. Genetic deletion of CARDI-B in mice from an isogenic background significantly increased the frequency of binucleated cardiomyocytes, at the expense of mononucleated cardiomyocytes. In line with previous reports that the frequency of mononucleated cardiomyocytes is correlated with cardiac regeneration, we show that CARDI-B-deficient hearts perform worse following myocardial infarction injury. These data reveal that functional micropeptides can be concealed within mammalian retroelements, and suggests that RE-derived micropeptides may contribute to the natural variation in heart development and regeneration observed across species.