Alternative 3'UTR expression induced by T cell activation is regulated in a temporal and signal dependent manner.

The length of 3' untranslated regions (3'UTR) is highly regulated during many transitions in cell state, including T cell activation, through the process of alternative polyadenylation (APA). However, the regulatory mechanisms and functional consequences of APA remain largely unexplored. Here we present a detailed analysis of the temporal and condition-specific regulation of APA following activation of primary human CD4+ T cells. We find that global APA changes are regulated temporally and CD28 costimulatory signals enhance a subset of these changes. Most APA changes upon T cell activation involve 3'UTR shortening, although a set of genes enriched for function in the mTOR pathway exhibit 3'UTR lengthening. While upregulation of the core polyadenylation machinery likely induces 3'UTR shortening following prolonged T cell stimulation; a significant program of APA changes occur prior to cellular proliferation or upregulation of the APA machinery. Motif analysis suggests that at least a subset of these early changes in APA are driven by upregulation of RBM3, an RNA-binding protein which competes with the APA machinery for binding. Together this work expands our understanding of the impact and mechanisms of APA in response to T cell activation and suggests new mechanisms by which APA may be regulated.

MAPP unravels frequent co-regulation of splicing and polyadenylation by RNA-binding proteins and their dysregulation in cancer.

Maturation of eukaryotic pre-mRNAs via splicing and polyadenylation is modulated across cell types and conditions by a variety of RNA-binding proteins (RBPs). Although there exist over 1,500 RBPs in human cells, their binding motifs and functions still remain to be elucidated, especially in the complex environment of tissues and in the context of diseases. To overcome the lack of methods for the systematic and automated detection of sequence motif-guided pre-mRNA processing regulation from RNA sequencing (RNA-Seq) data we have developed MAPP (Motif Activity on Pre-mRNA Processing). Applying MAPP to RBP knock-down experiments reveals that many RBPs regulate both splicing and polyadenylation of nascent transcripts by acting on similar sequence motifs. MAPP not only infers these sequence motifs, but also unravels the position-dependent impact of the RBPs on pre-mRNA processing. Interestingly, all investigated RBPs that act on both splicing and 3' end processing exhibit a consistently repressive or activating effect on both processes, providing a first glimpse on the underlying mechanism. Applying MAPP to normal and malignant brain tissue samples unveils that the motifs bound by the PTBP1 and RBFOX RBPs coordinately drive the oncogenic splicing program active in glioblastomas demonstrating that MAPP paves the way for characterizing pre-mRNA processing regulators under physiological and pathological conditions.

Chromatin regulates alternative polyadenylation via the RNA polymerase II elongation rate.

The RNA polymerase II (Pol II) elongation rate influences poly(A) site selection, with slow and fast Pol II derivatives causing upstream and downstream shifts, respectively, in poly(A) site utilization. In yeast, depletion of either of the histone chaperones FACT or Spt6 causes an upstream shift of poly(A) site use that strongly resembles the poly(A) profiles of slow Pol II mutant strains. Like slow Pol II mutant strains, FACT- and Spt6-depleted cells exhibit Pol II processivity defects, indicating that both Spt6 and FACT stimulate the Pol II elongation rate. Poly(A) profiles of some genes show atypical downstream shifts; this subset of genes overlaps well for FACT- or Spt6-depleted strains but is different from the atypical genes in Pol II speed mutant strains. In contrast, depletion of histone H3 or H4 causes a downstream shift of poly(A) sites for most genes, indicating that nucleosomes inhibit the Pol II elongation rate in vivo. Thus, chromatin-based control of the Pol II elongation rate is a potential mechanism, distinct from direct effects on the cleavage/polyadenylation machinery, to regulate alternative polyadenylation in response to genetic or environmental changes.

HPV oncogenes expressed from only one of multiple integrated HPV DNA copies drive clonal cell expansion in cervical cancer.

The integration of HPV DNA into human chromosomes plays a pivotal role in the onset of papillomavirus-related cancers. HPV DNA integration often occurs by linearizing the viral DNA in the E1/E2 region, resulting in the loss of a critical viral early polyadenylation signal (PAS), which is essential for the polyadenylation of the E6E7 bicistronic transcripts and for the expression of the viral E6 and E7 oncogenes. Here, we provide compelling evidence that, despite the presence of numerous integrated viral DNA copies, virus-host fusion transcripts originate from only a single integrated HPV DNA in HPV16 and HPV18 cervical cancers and cervical cancer-derived cell lines. The host genomic elements neighboring the integrated HPV DNA are critical for the efficient expression of the viral oncogenes that leads to clonal cell expansion. The fusion RNAs that are produced use a host RNA polyadenylation signal downstream of the integration site, and almost all involve splicing to host sequences. In cell culture, siRNAs specifically targeting the host portion of the virus-host fusion transcripts effectively silenced viral E6 and E7 expression. This, in turn, inhibited cell growth and promoted cell senescence in HPV16+ CaSki and HPV18+ HeLa cells. Showing that HPV E6 and E7 expression from a single integration site is instrumental in clonal cell expansion sheds new light on the mechanisms of HPV-induced carcinogenesis and could be used for the development of precision medicine tailored to combat HPV-related malignancies. IMPORTANCE: Persistent oncogenic HPV infections lead to viral DNA integration into the human genome and the development of cervical, anogenital, and oropharyngeal cancers. The expression of the viral E6 and E7 oncogenes plays a key role in cell transformation and tumorigenesis. However, how E6 and E7 could be expressed from the integrated viral DNA which often lacks a viral polyadenylation signal in the cancer cells remains unknown. By analyzing the integrated HPV DNA sites and expressed HPV RNAs in cervical cancer tissues and cell lines, we show that HPV oncogenes are expressed from only one of multiple chromosomal HPV DNA integrated copies. A host polyadenylation signal downstream of the integrated viral DNA is used for polyadenylation and stabilization of the virus-host chimeric RNAs, making the oncogenic transcripts targetable by siRNAs. This observation provides further understanding of the tumorigenic mechanism of HPV integration and suggests possible therapeutic strategies for the development of precision medicine for HPV cancers.

Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens using a dual fluorescence readthrough reporter.

Messenger RNA precursors (pre-mRNA) generally undergo 3' end processing by cleavage and polyadenylation (CPA), which is specified by a polyadenylation site (PAS) and adjacent RNA sequences and regulated by a large variety of core and auxiliary CPA factors. To date, most of the human CPA factors have been discovered through biochemical and proteomic studies. However, genetic identification of the human CPA factors has been hampered by the lack of a reliable genome-wide screening method. We describe here a dual fluorescence readthrough reporter system with a PAS inserted between two fluorescent reporters. This system enables measurement of the efficiency of 3' end processing in living cells. Using this system in combination with a human genome-wide CRISPR/Cas9 library, we conducted a screen for CPA factors. The screens identified most components of the known core CPA complexes and other known CPA factors. The screens also identified CCNK/CDK12 as a potential core CPA factor, and RPRD1B as a CPA factor that binds RNA and regulates the release of RNA polymerase II at the 3' ends of genes. Thus, this dual fluorescence reporter coupled with CRISPR/Cas9 screens reliably identifies bona fide CPA factors and provides a platform for investigating the requirements for CPA in various contexts.

CPEB2 inhibits preeclampsia progression by regulating SSTR3 translation through polyadenylation.

AIMS: Trophoblast cell dysfunction is one of the important factors leading to preeclampsia (PE). Cytoplasmic polyadenylation element-binding 2 (CPEB2) has been found to be differentially expressed in PE patients, but whether it mediates PE process by regulating trophoblast cell function is unclear. METHODS: The expression of CPEB2 and somatostatin receptor 3 (SSTR3) was detected by quantitative real-time PCR, Western blot (WB) and immunofluorescence staining. Cell functions were analyzed by CCK-8 assay, EdU assay, flow cytometry and transwell assay. Epithelial-mesenchymal transition (EMT)-related protein levels were detected by WB. The interaction of CPEB2 and SSTR3 was confirmed by RIP assay, dual-luciferase reporter assay and PCR poly(A) tail assay. Animal experiments were performed to explore the effect of CPEB2 on PE progression in vivo, and the placental tissues of rat were used for H&E staining, immunohistochemical staining and TUNEL staining. RESULTS: CPEB2 was lowly expressed in PE patients. CPEB2 upregulation accelerated trophoblast cell proliferation, migration, invasion and EMT, while its knockdown had an opposite effect. CPEB2 bound to the CPE site in the 3'-UTR of SSTR3 mRNA to suppress SSTR3 translation through reducing poly(A) tails. Besides, SSTR3 overexpression suppressed trophoblast cell proliferation, migration, invasion and EMT, while its silencing accelerated trophoblast cell functions. However, these effects could be reversed by CPEB2 upregulation and knockdown, respectively. In vivo experiments, CPEB2 overexpression relieved histopathologic changes, inhibited apoptosis, promoted proliferation and enhanced EMT in the placenta of PE rat by decreasing SSTR3 expression. CONCLUSION: CPEB2 inhibited PE progression, which promoted trophoblast cell functions by inhibiting SSTR3 translation through polyadenylation.

A highly optimized human in vitro translation system.

In vitro translation is an important method for studying fundamental aspects of co- and post-translational gene regulation, as well as for protein expression in the laboratory and on an industrial scale. Here, by re-examining and improving a human in vitro translation system (HITS), we were able to develop a minimal system where only four components are needed to supplement human cell lysates. Functional characterization of our improved HITS revealed the synergistic effect of mRNA capping and polyadenylation. Furthermore, we found that mRNAs are translated with an efficiency equal to or higher than existing state-of-the-art mammalian in vitro translation systems. Lastly, we present an easy preparation procedure for cytoplasmic extracts from cultured HeLa cells, which can be performed in any cell culture laboratory. These methodological advances will allow HITSs to become a widespread tool in basic molecular biology research.

Poly(A) tale: From A to A; RNA polyadenylation in prokaryotes and eukaryotes.

Most eukaryotic mRNAs and different non-coding RNAs undergo a form of 3' end processing known as polyadenylation. Polyadenylation machinery is present in almost all organisms except few species. In bacteria, the machinery has evolved from PNPase, which adds heteropolymeric tails, to a poly(A)-specific polymerase. Differently, a complex machinery for accurate polyadenylation and several non-canonical poly(A) polymerases are developed in eukaryotes. The role of poly(A) tail has also evolved from serving as a degradative signal to a stabilizing modification that also regulates translation. In this review, we discuss poly(A) tail emergence in prokaryotes and its development into a stable, yet dynamic feature at the 3' end of mRNAs in eukaryotes. We also describe how appearance of novel poly(A) polymerases gives cells flexibility to shape poly(A) tail. We explain how poly(A) tail dynamics help regulate cognate RNA metabolism in a context-dependent manner, such as during oocyte maturation. Finally, we describe specific mRNAs in metazoans that bear stem-loops instead of poly(A) tails. We conclude with how recent discoveries about poly(A) tail can be applied to mRNA technology. This article is categorized under: RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Processing > 3' End Processing RNA Turnover and Surveillance > Regulation of RNA Stability.

Non-canonical isoforms of the mRNA polyadenylation factor WDR33 regulate STING-mediated immune responses.

The human WDR33 gene encodes three major isoforms. The canonical isoform WDR33v1 (V1) is a well-characterized nuclear mRNA polyadenylation factor, while the other two, WDR33v2 (V2) and WDR33v3 (V3), have not been studied. Here, we report that V2 and V3 are generated by alternative polyadenylation, and neither protein contains all seven WD (tryptophan-aspartic acid) repeats that characterize V1. Surprisingly, V2 and V3 are not polyadenylation factors but localize to the endoplasmic reticulum and interact with stimulator of interferon genes (STING), the immune factor that induces the cellular response to cytosolic double-stranded DNA. V2 suppresses interferon-ß induction by preventing STING disulfide oligomerization but promotes autophagy, likely by recruiting WIPI2 isoforms. V3, on the other hand, functions to increase STING protein levels. Our study has not only provided mechanistic insights into STING regulation but also revealed that protein isoforms can be functionally completely unrelated, indicating that alternative mRNA processing is a more powerful mechanism than previously appreciated.

High-Throughput Spectroscopic Analysis of mRNA Capping Level.

Eukaryotic mRNAs are characterized by terminal 5' cap structures and 3' polyadenylation sites, which are essential for posttranscriptional processing, translation initiation, and stability. Here, we describe a novel biosensor method designed to detect the presence of both cap structures and polyadenylation sites on mRNA molecules. This novel biosensor is sensitive to mRNA degradation and can quantitatively determine capping levels of mRNA molecules within a mixture of capped and uncapped mRNA molecules. The biosensor displays a constant dynamic range between 254 nt and 6507 nt with reproducible sensitivity to increases in capping level of at least 20% and a limit of detection of 2.4 pmol of mRNA. Overall, the biosensor can provide key information about mRNA quality before mammalian cell transfection.

Rational design of an artificial tethered enzyme for non-templated post-transcriptional mRNA polyadenylation by the second generation of the C3P3 system.

We have previously introduced the first generation of C3P3, an artificial system that allows the autonomous in-vivo production of mRNA with m7GpppN-cap. While C3P3-G1 synthesized much larger amounts of capped mRNA in human cells than conventional nuclear expression systems, it produced a proportionately much smaller amount of the corresponding proteins, indicating a clear defect of mRNA translatability. A possible mechanism for this poor translatability could be the rudimentary polyadenylation of the mRNA produced by the C3P3-G1 system. We therefore sought to develop the C3P3-G2 system using an artificial enzyme to post-transcriptionally lengthen the poly(A) tail. This system is based on the mutant mouse poly(A) polymerase alpha fused at its N terminus with an N peptide from the λ virus, which binds to BoxBr sequences placed in the 3'UTR region of the mRNA of interest. The resulting system selectively brings mPAPαm7 to the target mRNA to elongate its poly(A)-tail to a length of few hundred adenosine. Such elongation of the poly(A) tail leads to an increase in protein expression levels of about 2.5-3 times in cultured human cells compared to the C3P3-G1 system. Finally, the coding sequence of the tethered mutant poly(A) polymerase can be efficiently fused to that of the C3P3-G1 enzyme via an F2A sequence, thus constituting the single-ORF C3P3-G2 enzyme. These technical developments constitute an important milestone in improving the performance of the C3P3 system, paving the way for its applications in bioproduction and non-viral human gene therapy.

ASAPA: a bioinformatic pipeline based on Iso-Seq that identifies the links among alternative splicing, alternative transcription initiation and alternative polyadenylation.

BACKGROUND: Although the events associated with alternative splicing (AS), alternative polyadenylation (APA) and alternative transcription initiation (ATI) can be identified by many approaches based on isoform sequencing (Iso-Seq), these analyses are generally independent of each other and the links between these events are still rarely mentioned. However, an interdependency analysis can be achieved because the transcriptional start site, splice sites and polyA site could be simultaneously included in a long, full-length read from Iso-Seq. RESULTS: We create ASAPA pipeline that enables streamlined analysis for a robust detection of potential links among AS, ATI and APA using Iso-Seq data. We tested this pipeline using Arabidopsis data and found some interesting results: some adjacent introns tend to be simultaneously spliced or retained; coupling between AS and ATI or APA is limited to the initial or terminal intron; and ATI and APA are potentially linked in some special cases. CONCLUSION: Our pipeline enables streamlined analysis for a robust detection of potential links among AS, ATI and APA using Iso-Seq data, which is conducive to a better understanding of transcription landscape generation.

The Transcriptional Landscape of Immune-Response 3'-UTR Alternative Polyadenylation in Melanoma.

The prognosis of patients with malignant melanoma has been improved in recent decades due to advancements in immunotherapy. However, a considerable proportion of patients are refractory to treatment, particularly at advanced stages. This underscores the necessity of developing a new strategy to improve it. Alternative polyadenylation (APA), as a marker of crucial posttranscriptional regulation, has emerged as a major new type of epigenetic marker involved in tumorigenesis. However, the potential roles of APA in shaping the tumor microenvironment (TME) are largely unexplored. Herein, we collected two cohorts comprising melanoma patients who received immune checkpoint inhibitor (ICI) immunotherapy to quantify transcriptome-wide discrepancies in APA. We observed a global change in 3'-UTRs between responders and non-responders, which might involve DNA damage response, angiogenesis, PI3K-AKT signaling pathways, etc. Ten putative master APA regulatory factors for those APA events were detected via a network analysis. Notably, we established an immune response-related APA scoring system (IRAPAss), which exhibited a great performance of predicting immunotherapy response in multiple cohorts. Furthermore, we examined the correlation of APA with TME at the single-cell level using four single-cell immune profiles of tumor-infiltrating lymphocytes (TILs), which revealed an overall discrepancy in 3'-UTR length across diverse T cell populations, probably contributing to immunoregulation in melanoma. In conclusion, our study provides a transcriptional landscape of APA implicated in immunoregulation, which might lay the foundation for developing a new strategy for improving immunotherapy response for melanoma patients by targeting APA.

Control of poly(A)-tail length and translation in vertebrate oocytes and early embryos.

During oocyte maturation and early embryogenesis, changes in mRNA poly(A)-tail lengths strongly influence translation, but how these tail-length changes are orchestrated has been unclear. Here, we performed tail-length and translational profiling of mRNA reporter libraries (each with millions of 3' UTR sequence variants) in frog oocytes and embryos and in fish embryos. Contrasting to previously proposed cytoplasmic polyadenylation elements (CPEs), we found that a shorter element, UUUUA, together with the polyadenylation signal (PAS), specify cytoplasmic polyadenylation, and we identified contextual features that modulate the activity of both elements. In maturing oocytes, this tail lengthening occurs against a backdrop of global deadenylation and the action of C-rich elements that specify tail-length-independent translational repression. In embryos, cytoplasmic polyadenylation becomes more permissive, and additional elements specify waves of stage-specific deadenylation. Together, these findings largely explain the complex tapestry of tail-length changes observed in early frog and fish development, with strong evidence of conservation in both mice and humans.

InPACT: a computational method for accurate characterization of intronic polyadenylation from RNA sequencing data.

Alternative polyadenylation can occur in introns, termed intronic polyadenylation (IPA), has been implicated in diverse biological processes and diseases, as it can produce noncoding transcripts or transcripts with truncated coding regions. However, a reliable method is required to accurately characterize IPA. Here, we propose a computational method called InPACT, which allows for the precise characterization of IPA from conventional RNA-seq data. InPACT successfully identifies numerous previously unannotated IPA transcripts in human cells, many of which are translated, as evidenced by ribosome profiling data. We have demonstrated that InPACT outperforms other methods in terms of IPA identification and quantification. Moreover, InPACT applied to monocyte activation reveals temporally coordinated IPA events. Further application on single-cell RNA-seq data of human fetal bone marrow reveals the expression of several IPA isoforms in a context-specific manner. Therefore, InPACT represents a powerful tool for the accurate characterization of IPA from RNA-seq data.

vizAPA: visualizing dynamics of alternative polyadenylation from bulk and single-cell data.

MOTIVATION: Alternative polyadenylation (APA) is a widespread post-transcriptional regulatory mechanism across all eukaryotes. With the accumulation of genome-wide APA sites, especially those with single-cell resolution, it is imperative to develop easy-to-use visualization tools to guide APA analysis. RESULTS: We developed an R package called vizAPA for visualizing APA dynamics from bulk and single-cell data. vizAPA implements unified data structures for APA data and genome annotations. vizAPA also enables identification of genes with differential APA usage across biological samples and/or cell types. vizAPA provides four unique modules for extensively visualizing APA dynamics across biological samples and at the single-cell level. vizAPA could serve as a plugin in many routine APA analysis pipelines to augment studies for APA dynamics. AVAILABILITY AND IMPLEMENTATION: https://github.com/BMILAB/vizAPA.

Downregulation of CPSF6 leads to global mRNA 3' UTR shortening and enhanced antiviral immune responses.

Alternative polyadenylation (APA) is a widespread mechanism of gene regulation that generates mRNA isoforms with alternative 3' untranslated regions (3' UTRs). Our previous study has revealed the global 3' UTR shortening of host mRNAs through APA upon viral infection. However, how the dynamic changes in the APA landscape occur upon viral infection remains largely unknown. Here we further found that, the reduced protein abundance of CPSF6, one of the core 3' processing factors, promotes the usage of proximal poly(A) sites (pPASs) of many immune related genes in macrophages and fibroblasts upon viral infection. Shortening of the 3' UTR of these transcripts may improve their mRNA stability and translation efficiency, leading to the promotion of type I IFN (IFN-I) signalling-based antiviral immune responses. In addition, dysregulated expression of CPSF6 is also observed in many immune related physiological and pathological conditions, especially in various infections and cancers. Thus, the global APA dynamics of immune genes regulated by CPSF6, can fine-tune the antiviral response as well as the responses to other cellular stresses to maintain the tissue homeostasis, which may represent a novel regulatory mechanism for antiviral immunity.

T helper cells exhibit a dynamic and reversible 3'-UTR landscape.

3' untranslated regions (3' UTRs) are critical elements of messenger RNAs, as they contain binding sites for RNA-binding proteins (RBPs) and microRNAs that affect various aspects of the RNA life cycle including transcript stability and cellular localization. In response to T cell receptor activation, T cells undergo massive expansion during the effector phase of the immune response and dynamically modify their 3' UTRs. Whether this serves to directly regulate the abundance of specific mRNAs or is a secondary effect of proliferation remains unclear. To study 3'-UTR dynamics in T helper cells, we investigated division-dependent alternative polyadenylation (APA). In addition, we generated 3' end UTR sequencing data from naive, activated, memory, and regulatory CD4+ T cells. 3'-UTR length changes were estimated using a nonnegative matrix factorization approach and were compared with those inferred from long-read PacBio sequencing. We found that APA events were transient and reverted after effector phase expansion. Using an orthogonal bulk RNA-seq data set, we did not find evidence of APA association with differential gene expression or transcript usage, indicating that APA has only a marginal effect on transcript abundance. 3'-UTR sequence analysis revealed conserved binding sites for T cell-relevant microRNAs and RBPs in the alternative 3' UTRs. These results indicate that poly(A) site usage could play an important role in the control of cell fate decisions and homeostasis.

Circulating tumor cell clustering modulates RNA splicing and polyadenylation to facilitate metastasis.

Circulating tumor cell (CTC) clusters exhibit significantly higher metastatic potential compared to single CTCs. However, the underlying mechanism behind this phenomenon remains unclear, and the role of posttranscriptional RNA regulation in CTC clusters has not been explored. Here, we conducted a comparative analysis of alternative splicing (AS) and alternative polyadenylation (APA) profiles between single CTCs and CTC clusters. We identified 994 and 836 AS events in single CTCs and CTC clusters, respectively, with ∼20% of AS events showing differential regulation between the two cell types. A key event in this differential splicing was observed in SRSF6, which disrupted AS profiles and contributed to the increased malignancy of CTC clusters. Regarding APA, we found a global lengthening of 3' UTRs in CTC clusters compared to single CTCs. This alteration was primarily governed by 14 core APA factors, particularly PPP1CA. The modified APA profiles facilitated the cell cycle progression of CTC clusters and indicated their reduced susceptibility to oxidative stress. Further investigation revealed that the proportion of H2AFY mRNA with long 3' UTR instead of short 3' UTR was higher in CTC clusters than single CTCs. The AU-rich elements (AREs) within the long 3' UTR of H2AFY mRNA enhance mRNA stability and translation activity, resulting in promoting cell proliferation and invasion, which potentially facilitate the establishment and rapid formation of metastatic tumors mediated by CTC clusters. These findings provide new insights into the mechanisms driving CTC cluster metastasis.

Inhibition of Cpeb3 ribozyme elevates CPEB3 protein expression and polyadenylation of its target mRNAs and enhances object location memory.

A self-cleaving ribozyme that maps to an intron of the cytoplasmic polyadenylation element-binding protein 3 (Cpeb3) gene is thought to play a role in human episodic memory, but the underlying mechanisms mediating this effect are not known. We tested the activity of the murine sequence and found that the ribozyme's self-scission half-life matches the time it takes an RNA polymerase to reach the immediate downstream exon, suggesting that the ribozyme-dependent intron cleavage is tuned to co-transcriptional splicing of the Cpeb3 mRNA. Our studies also reveal that the murine ribozyme modulates maturation of its harboring mRNA in both cultured cortical neurons and the hippocampus: inhibition of the ribozyme using an antisense oligonucleotide leads to increased CPEB3 protein expression, which enhances polyadenylation and translation of localized plasticity-related target mRNAs, and subsequently strengthens hippocampal-dependent long-term memory. These findings reveal a previously unknown role for self-cleaving ribozyme activity in regulating experience-induced co-transcriptional and local translational processes required for learning and memory.

Stored within DNA are the instructions cells need to make proteins. In order for proteins to get made, the region of DNA that codes for the desired protein (known as the gene) must first be copied into a molecule called messenger RNA (or mRNA for short). Once transcribed, the mRNA undergoes further modifications, including removing redundant segments known as introns. It then travels to molecular machines that translate its genetic sequence into the building blocks of the protein. Following transcription, some RNAs can fold into catalytic segments known as self-cleaving ribozymes which promote the scission of their own genetic sequence. One such ribozyme resides in the intron of a gene for CPEB3, a protein which adds a poly(A) tail to various mRNAs, including some involved in learning and memory. Although this ribozyme is found in most mammals, its biological role is poorly understood. Previous studies suggested that the ribozyme cleaves itself at the same time as the mRNA for CPEB3 is transcribed. This led Chen et al. to hypothesize that the rate at which these two events occur impacts the amount of CPEB3 produced, resulting in changes in memory and learning. If the ribozyme cleaves quickly, the intron is disrupted and may not be properly removed, leading to less CPEB3 being made. However, if the ribozyme is inhibited, the intron remains intact and is efficiently excised, resulting in higher levels of CPEB3 protein. To test how the ribozyme impacts CPEB3 production, Chen et al. inhibited the enzyme from cutting itself with antisense oligonucleotides (ASOs). The ASOs were applied to in vitro transcription systems, neurons cultured in the laboratory and the brains of living mice in an area called the hippocampus. The in vitro and cell culture experiments led to higher levels of CPEB3 protein and the addition of more poly(A) tails to mRNAs involved in neuron communication. Injection of the ASOs into the brains of mice had the same effect, and also improved their memory and learning. The findings of Chen et al. show a new mechanism for controlling protein production, and suggest that ASOs could be used to increase the levels of CPEB3 and modulate neuronal activity. This is the first time a biological role for a self-cleaving ribozyme in mammals has been identified, and the approach used could be applied to investigate the function of two other self-cleaving ribozymes located in introns in humans.