The Role of Alternative Polyadenylation in the Regulation of Subcellular RNA Localization

Alternative polyadenylation (APA) is a widespread and conserved regulatory mechanism that generates diverse 3′ ends on mRNA. APA patterns are often tissue specific and play an important role in cellular processes such as cell proliferation, differentiation, and response to stress. Many APA sites are found in 3′ UTRs, generating mRNA isoforms with different 3′ UTR contents. These alternate 3′ UTR isoforms can change how the transcript is regulated, affecting its stability and translation. Since the subcellular localization of a transcript is often regulated by 3′ UTR sequences, this implies that APA can also change transcript location. However, this connection between APA and RNA localization has only recently been explored. In this review, we discuss the role of APA in mRNA localization across distinct subcellular compartments. We also discuss current challenges and future advancements that will aid our understanding of how APA affects RNA localization and molecular mechanisms that drive these processes.

Pax-5 Protein Expression Is Regulated by Transcriptional 3′UTR Editing

The Pax-5 gene encodes a transcription factor that is essential for B-cell commitment and maturation. However, Pax-5 deregulation is associated with various cancer lesions, notably hematopoietic cancers. Mechanistically, studies have characterized genetic alterations within the Pax-5 locus that result in either dominant oncogenic function or haploinsufficiency-inducing mutations leading to oncogenesis. Apart from these mutations, some examples of aberrant Pax-5 expression cannot be associated with genetic alterations. In the present study, we set out to elucidate potential alterations in post-transcriptional regulation of Pax-5 expression and establish that Pax-5 transcript editing represents an important means to aberrant expression. Upon the profiling of Pax-5 mRNA in leukemic cells, we found that the 3′end of the Pax-5 transcript is submitted to alternative polyadenylation (APA) and alternative splicing events. Using rapid amplification of cDNA ends (3′RACE) from polysomal fractions, we found that Pax-5 3′ untranslated region (UTR) shortening correlates with increased ribosomal occupancy for translation. These observations were also validated using reporter gene assays with truncated 3′UTR regions cloned downstream of a luciferase gene. We also showed that Pax-5 3′UTR editing has direct repercussions on regulatory elements such as miRNAs, which in turn impact Pax-5 protein expression. More importantly, we found that advanced staging of various hematopoietic cancer lesions relates to shorter Pax-5 3′UTRs. Altogether, our findings identify novel molecular mechanisms that account for aberrant expression and function of the Pax-5 oncogene in cancer cells. These findings also present new avenues for strategic intervention in Pax-5-mediated cancers.

FMRP regulates mRNAs encoding distinct functions in the cell body and dendrites of CA1 pyramidal neurons

Neurons rely on translation of synaptic mRNAs in order to generate activity-dependent changes in plasticity. Here we develop a strategy combining compartment-specific CLIP and TRAP in conditionally tagged mice to precisely define the ribosome-bound dendritic transcriptome of CA1 pyramidal neurons. We identify CA1 dendritic transcripts with differentially localized mRNA isoforms generated by alternative polyadenylation and alternative splicing, including many which have altered protein-coding capacity. Among dendritic mRNAs, FMRP targets were found to be overrepresented. Cell-type specific FMRP-CLIP and TRAP in microdissected CA1 neuropil revealed 383 dendritic FMRP targets and suggests that FMRP differentially regulates functionally distinct modules in CA1 dendrites and cell bodies. FMRP regulates ~15-20% of mRNAs encoding synaptic functions and 10% of chromatin modulators, in the dendrite and cell body, respectively. In the absence of FMRP, dendritic FMRP targets had increased ribosome association, consistent with a function for FMRP in synaptic translational repression. Conversely, downregulation of FMRP targets involved in chromatin regulation in cell bodies and suggest a role for FMRP in stabilizing mRNAs containing stalled ribosomes in this compartment. Together, the data support a model in which FMRP regulates the translation and expression of synaptic and nuclear proteins within different compartments of a single neuronal cell type.

CFIm25 regulates human stem cell function independently of its role in mRNA alternative polyadenylation

It has recently been shown that CFIm25, a canonical mRNA 3’ processing factor, could play a variety of physiological roles through its molecular function in the regulation of mRNA alternative polyadenylation (APA). Here, we used CRISPR/Cas9-mediated gene editing approach in human embryonic stem cells (hESCs) for CFIm25, and obtained three gene knockdown/mutant cell lines. CFIm25 gene editing resulted in higher proliferation rate and impaired differentiation potential for hESCs, with these effects likely to be directly regulated by the target genes, including the pluripotency factor rex1. Mechanistically, we unexpected found that perturbation in CFIm25 gene expression did not significantly affect cellular mRNA 3’ processing efficiency and APA profile. Rather, we provided evidences that CFIm25 may impact RNA polymerase II (RNAPII) occupancy at the body of transcribed genes, and promote the expression level of a group of transcripts associated with cellular proliferation and/or differentiation. Further study indicated that CFIm25 association with LEO1, an RNAPII associated factor, might contribute to the effect. Taken together, these results reveal novel mechanisms underlying CFIm25’s modulation in determination of cell fate, and provide evidence that the process of mammalian gene transcription may be regulated by an mRNA 3’ processing factor.

Specific alternative splicing and polyadenylation facilitate metastasis mediated by CTC clusters

Circulating tumor cell (CTC) clusters possess a much higher capability to seed metastasis than single CTCs. However, the mechanism underlying this phenomenon is still elusive and no reports have investigated the role of posttranscriptional RNA regulation in CTC clusters. Here, we compared alternative splicing (AS) and alternative polyadenylation (APA) profiles between single CTCs and CTC clusters. 994 and 836 AS events were identified in single CTCs and CTC clusters, separately. About ~20% of AS events exhibited alterations between both cell types. The differential splicing of SRSF6 was a core event that caused AS profiles’ disturbance and made CTC clusters more dangerous. Concerning APA, we identified global 3’ UTRs lengthening in CTC clusters compared with single CTCs. This change was mainly regulated by 14 core APA factors, especially PPP1CA. The altered APA profiles boosted the cell cycle of CTC clusters and reflected that CTC clusters endured less oxidative stress. Our study investigated the posttranscriptional regulation mechanisms in CTC clusters, found that the perturbation of AS and APA contributed to the superiority of CTC clusters compared with single CTCs, and laid the foundation for developing antisense oligonucleotides that inhibit metastasis by reducing CTC clusters.

Tau Modulates mRNA Transcription, Alternative Polyadenylation Profiles of hnRNPs, Chromatin Remodeling and Spliceosome Complexes

Tau protein is a known contributor in several neurodegenerative diseases, including Alzheimer’s disease (AD) and frontotemporal dementia (FTD). It is well-established that tau forms pathological aggregates and fibrils in these diseases. Tau has been observed within the nuclei of neurons, but there is a gap in understanding regarding the mechanism by which tau modulates transcription. We are interested in the P301L mutation of tau, which has been associated with FTD and increased tau aggregation. Our study utilized tau-inducible HEK (iHEK) cells to reveal that WT and P301L tau distinctively alter the transcription and alternative polyadenylation (APA) profiles of numerous nuclear precursors mRNAs, which then translate to form proteins involved in chromatin remodeling and splicing. We isolated total mRNA before and after over-expressing tau and then performed Poly(A)-ClickSeq (PAC-Seq) to characterize mRNA expression and APA profiles. We characterized changes in Gene Ontology (GO) pathways using EnrichR and Gene Set Enrichment Analysis (GSEA). We observed that P301L tau up-regulates genes associated with reactive oxygen species responsiveness as well as genes involved in dendrite, microtubule, and nuclear body/speckle formation. The number of genes regulated by WT tau is greater than the mutant form, which indicates that the P301L mutation causes loss-of-function at the transcriptional level. WT tau up-regulates genes contributing to cytoskeleton-dependent intracellular transport, microglial activation, microtubule and nuclear chromatin organization, formation of nuclear bodies and speckles. Interestingly, both WT and P301L tau commonly down-regulate genes responsible for ubiquitin-proteosome system. In addition, WT tau significantly down-regulates several genes implicated in chromatin remodeling and nucleosome organization. Although there are limitations inherent to the model systems used, this study will improve understanding regarding the nuclear impact of tau at the transcriptional and post-transcriptional level. This study also illustrates the potential impact of P301L tau on the human brain genome during early phases of pathogenesis.

Comprehensive mapping of the alternative polyadenylation site usage and its dynamics at single cell resolution

Alternative polyadenylation (APA) plays an important role in post-transcriptional gene regulation such as transcript stability and translation efficiency. However, our knowledge about APA dynamics at single cell level is largely unexplored. Here we developed single cell polyadenylation sequencing (scPolyA-seq), a strand-specific approach for sequencing 3’ end of transcripts, to investigate the landscape of APA at single cell level. By analyzing several cell lines, we found many genes using multiple polyA sites in bulk data are prone to use only one polyA site in each single cell. Interestingly, cell cycle was significantly enriched in genes showing high variation of polyA site usages. We further identified 414 genes showing polyA site usage switch after cell synchronization. Genes showing cell cycle associated polyA site usage switch were grouped into 6 clusters, with cell phase specific functional categories enriched in each cluster. Furthermore, scPolyA-seq could facilitate study of APA in various biological processes.

Alternative Polyadenylation Associated with Prognosis and Therapy in Colorectal Cancer

Colorectal cancer (CRC) is among the most widely spread cancers globally. Aberrant alternative polyadenylation (APA) plays a role in cancer onset and its progression. Consequently, this study focused on highlighting the role of APA events and signals in the prognosis of patients with CRC. The APA events, RNA sequencing (RNA-seq), somatic mutations, copy number variants (CNVs), and clinical information of the CRC cohort were obtained from The Cancer Genome Atlas (TCGA) database and UCSC (University of California-Santa Cruz) Xena database. The entire set was sorted into two sets: a training set and a test set in a ratio of 1:1. 197 prognosis-related APA events were collected by performing univariate Cox regression signature in patients with CRC. Subsequently, a signature for APA events was established by least absolute shrinkage and selection operator (LASSO) and multivariate Cox analysis. The risk scores were measured for individual patients on the basis of the signature and patients were sorted into two groups; the high-risk group and the low-risk group as per their median risk scores. Kaplan-Meier curves, principal component analysis (PCA), and time-dependent receiver operator characteristic (ROC) curves revealed that the signature was able to predict patient prognosis effectively and further validation was provided in the test set and the entire set. Both the groups highlighted various distributions of mutations and CNVs. Tumor mutation burden (TMB) alone and in combination with the signature predicted the prognosis of CRC patients, but the gene frequencies of TMBs and CNVs did not change in the low- and high-risk groups. Moreover, immunotherapy and chemotherapy treatments showed different responses to PD-1 inhibitors and 26 chemotherapeutic agents in the low and high-risk groups based on the tumor immune dysfunction and exclusion (TIDE) and genomics of drugs sensitivity in cancer (GDSC) databases. This study helped in understanding APA events during the progression of CRC in great detail, and the signature for prognosis-related APA events can work as the potential predictors for survival and treatment in patients with CRC.