Neuronal Cells Display Distinct Stability Controls of Alternative Polyadenylation mRNA Isoforms, Long Non-Coding RNAs, and Mitochondrial RNAs.

RNA stability plays an important role in gene expression. Here, using 3' end sequencing of newly made and pre-existing poly(A)+ RNAs, we compare transcript stability in multiple human cell lines, including HEK293T, HepG2, and SH-SY5Y. We show that while mRNA stability is generally conserved across the cell lines, specific transcripts having a high GC content and possibly more stable secondary RNA structures are relatively more stable in SH-SY5Y cells compared to the other 2 cell lines. These features also differentiate stability levels of alternative polyadenylation (APA) 3'UTR isoforms in a cell type-specific manner. Using differentiation of a neural stem cell line as a model, we show that mRNA stability difference could contribute to gene expression changes in neurogenesis and confirm the neuronal identity of SH-SY5Y cells at both gene expression and APA levels. In addition, compared to transcripts using 3'-most exon cleavage/polyadenylation sites (PASs), those using intronic PASs are generally less stable, especially when the PAS-containing intron is large and has a strong 5' splice site, suggesting that intronic polyadenylation mostly plays a negative role in gene expression. Interestingly, the differential mRNA stability among APA isoforms appears to buffer PAS choice in these cell lines. Moreover, we found that several other poly(A)+ RNA species, including promoter-associated long noncoding RNAs and transcripts encoded by the mitochondrial genome, are more stable in SH-SY5Y cells than the other 2 cell lines, further highlighting distinct RNA metabolism in neuronal cells. Together, our results indicate that distinct RNA stability control in neuronal cells may contribute to the gene expression and APA programs that define their cell identity.

ZFC3H1 and U1-70K promote the nuclear retention of mRNAs with 5' splice site motifs within nuclear speckles.

Quality control of mRNA represents an important regulatory mechanism for gene expression in eukaryotes. One component of this quality control is the nuclear retention and decay of misprocessed RNAs. Previously, we demonstrated that mature mRNAs containing a 5' splice site (5'SS) motif, which is typically found in misprocessed RNAs such as intronic polyadenylated (IPA) transcripts, are nuclear retained and degraded. Using high-throughput sequencing of cellular fractions, we now demonstrate that IPA transcripts require the zinc finger protein ZFC3H1 for their nuclear retention and degradation. Using reporter mRNAs, we demonstrate that ZFC3H1 promotes the nuclear retention of mRNAs with intact 5'SS motifs by sequestering them into nuclear speckles. Furthermore, we find that U1-70K, a component of the spliceosomal U1 snRNP, is also required for the nuclear retention of these reporter mRNAs and likely functions in the same pathway as ZFC3H1. Finally, we show that the disassembly of nuclear speckles impairs the nuclear retention of reporter mRNAs with 5'SS motifs. Our results highlight a splicing independent role of U1 snRNP and indicate that it works in conjunction with ZFC3H1 in preventing the nuclear export of misprocessed mRNAs by sequestering them into nuclear speckles.

CRISPRpas: programmable regulation of alternative polyadenylation by dCas9.

Most human protein-coding genes produce alternative polyadenylation (APA) isoforms that differ in 3' UTR size or, when coupled with splicing, have variable coding sequences. APA is an important layer of gene expression program critical for defining cell identity. Here, by using a catalytically dead Cas9 and coupling its target site with polyadenylation site (PAS), we develop a method, named CRISPRpas, to alter APA isoform abundance. CRISPRpas functions by enhancing proximal PAS usage, whose efficiency is influenced by several factors, including targeting strand of DNA, distance between PAS and target sequence and strength of the PAS. For intronic polyadenylation (IPA), splicing features, such as strengths of 5' splice site and 3' splice site, also affect CRISPRpas efficiency. We show modulation of APA of multiple endogenous genes, including IPA of PCF11, a master regulator of APA and gene expression. In sum, CRISPRpas offers a programmable tool for APA regulation that impacts gene expression.

Alternative 3' UTRs play a widespread role in translation-independent mRNA association with the endoplasmic reticulum.

Transcripts encoding membrane and secreted proteins are known to associate with the endoplasmic reticulum (ER) through translation. Here, using cell fractionation, polysome profiling, and 3' end sequencing, we show that transcripts differ substantially in translation-independent ER association (TiERA). Genes in certain functional groups, such as cell signaling, tend to have significantly higher TiERA potentials than others, suggesting the importance of ER association for their mRNA metabolism, such as localized translation. The TiERA potential of a transcript is determined largely by size, sequence content, and RNA structures. Alternative polyadenylation (APA) isoforms can have distinct TiERA potentials because of changes in transcript features. The widespread 3' UTR lengthening in cell differentiation leads to greater transcript association with the ER, especially for genes that are capable of expressing very long 3' UTRs. Our data also indicate that TiERA is in dynamic competition with translation-dependent ER association, suggesting limited space on the ER for mRNA association.

Analysis of alternative cleavage and polyadenylation in mature and differentiating neurons using RNA-seq data.

BACKGROUND: Most eukaryotic protein-coding genes exhibit alternative cleavage and polyadenylation (APA), resulting in mRNA isoforms with different 3' untranslated regions (3' UTRs). Studies have shown that brain cells tend to express long 3' UTR isoforms using distal cleavage and polyadenylation sites (PASs). METHODS: Using our recently developed, comprehensive PAS database PolyA_DB, we developed an efficient method to examine APA, named Significance Analysis of Alternative Polyadenylation using RNA-seq (SAAP-RS). We applied this method to study APA in brain cells and neurogenesis. RESULTS: We found that neurons globally express longer 3' UTRs than other cell types in brain, and microglia and endothelial cells express substantially shorter 3' UTRs. We show that the 3' UTR diversity across brain cells can be corroborated with single cell sequencing data. Further analysis of APA regulation of 3' UTRs during differentiation of embryonic stem cells into neurons indicates that a large fraction of the APA events regulated in neurogenesis are similarly modulated in myogenesis, but to a much greater extent. CONCLUSION: Together, our data delineate APA profiles in different brain cells and indicate that APA regulation in neurogenesis is largely an augmented process taking place in other types of cell differentiation.

Activity-Dependent Regulation of Alternative Cleavage and Polyadenylation During Hippocampal Long-Term Potentiation.

Long-lasting forms of synaptic plasticity that underlie learning and memory require new transcription and translation for their persistence. The remarkable polarity and compartmentalization of neurons raises questions about the spatial and temporal regulation of gene expression within neurons. Alternative cleavage and polyadenylation (APA) generates mRNA isoforms with different 3' untranslated regions (3'UTRs) and/or coding sequences. Changes in the 3'UTR composition of mRNAs can alter gene expression by regulating transcript localization, stability and/or translation, while changes in the coding sequences lead to mRNAs encoding distinct proteins. Using specialized 3' end deep sequencing methods, we undertook a comprehensive analysis of APA following induction of long-term potentiation (LTP) of mouse hippocampal CA3-CA1 synapses. We identified extensive LTP-induced APA changes, including a general trend of 3'UTR shortening and activation of intronic APA isoforms. Comparison with transcriptome profiling indicated that most APA regulatory events were uncoupled from changes in transcript abundance. We further show that specific APA regulatory events can impact expression of two molecules with known functions during LTP, including 3'UTR APA of Notch1 and intronic APA of Creb1. Together, our results reveal that activity-dependent APA provides an important layer of gene regulation during learning and memory.

Quantifying the Determinants of Evolutionary Dynamics Leading to Drug Resistance.

The emergence of drug resistant pathogens is a serious public health problem. It is a long-standing goal to predict rates of resistance evolution and design optimal treatment strategies accordingly. To this end, it is crucial to reveal the underlying causes of drug-specific differences in the evolutionary dynamics leading to resistance. However, it remains largely unknown why the rates of resistance evolution via spontaneous mutations and the diversity of mutational paths vary substantially between drugs. Here we comprehensively quantify the distribution of fitness effects (DFE) of mutations, a key determinant of evolutionary dynamics, in the presence of eight antibiotics representing the main modes of action. Using precise high-throughput fitness measurements for genome-wide Escherichia coli gene deletion strains, we find that the width of the DFE varies dramatically between antibiotics and, contrary to conventional wisdom, for some drugs the DFE width is lower than in the absence of stress. We show that this previously underappreciated divergence in DFE width among antibiotics is largely caused by their distinct drug-specific dose-response characteristics. Unlike the DFE, the magnitude of the changes in tolerated drug concentration resulting from genome-wide mutations is similar for most drugs but exceptionally small for the antibiotic nitrofurantoin, i.e., mutations generally have considerably smaller resistance effects for nitrofurantoin than for other drugs. A population genetics model predicts that resistance evolution for drugs with this property is severely limited and confined to reproducible mutational paths. We tested this prediction in laboratory evolution experiments using the "morbidostat", a device for evolving bacteria in well-controlled drug environments. Nitrofurantoin resistance indeed evolved extremely slowly via reproducible mutations-an almost paradoxical behavior since this drug causes DNA damage and increases the mutation rate. Overall, we identified novel quantitative characteristics of the evolutionary landscape that provide the conceptual foundation for predicting the dynamics of drug resistance evolution.

Strength of selection pressure is an important parameter contributing to the complexity of antibiotic resistance evolution.

Revealing the genetic changes responsible for antibiotic resistance can be critical for developing novel antibiotic therapies. However, systematic studies correlating genotype to phenotype in the context of antibiotic resistance have been missing. In order to fill in this gap, we evolved 88 isogenic Escherichia coli populations against 22 antibiotics for 3 weeks. For every drug, two populations were evolved under strong selection and two populations were evolved under mild selection. By quantifying evolved populations' resistances against all 22 drugs, we constructed two separate cross-resistance networks for strongly and mildly selected populations. Subsequently, we sequenced representative colonies isolated from evolved populations for revealing the genetic basis for novel phenotypes. Bacterial populations that evolved resistance against antibiotics under strong selection acquired high levels of cross-resistance against several antibiotics, whereas other bacterial populations evolved under milder selection acquired relatively weaker cross-resistance. In addition, we found that strongly selected strains against aminoglycosides became more susceptible to five other drug classes compared with their wild-type ancestor as a result of a point mutation on TrkH, an ion transporter protein. Our findings suggest that selection strength is an important parameter contributing to the complexity of antibiotic resistance problem and use of high doses of antibiotics to clear infections has the potential to promote increase of cross-resistance in clinics.

Large-scale identification and analysis of suppressive drug interactions.

One drug may suppress the effects of another. Although knowledge of drug suppression is vital to avoid efficacy-reducing drug interactions or discover countermeasures for chemical toxins, drug-drug suppression relationships have not been systematically mapped. Here, we analyze the growth response of Saccharomyces cerevisiae to anti-fungal compound ("drug") pairs. Among 440 ordered drug pairs, we identified 94 suppressive drug interactions. Using only pairs not selected on the basis of their suppression behavior, we provide an estimate of the prevalence of suppressive interactions between anti-fungal compounds as 17%. Analysis of the drug suppression network suggested that Bromopyruvate is a frequently suppressive drug and Staurosporine is a frequently suppressed drug. We investigated potential explanations for suppressive drug interactions, including chemogenomic analysis, coaggregation, and pH effects, allowing us to explain the interaction tendencies of Bromopyruvate.