Sequence and tissue targeting specificity of ZFP36L2 reveals Elavl2 as a novel target with co-regulation potential.

Zinc finger protein 36 like 2 (ZFP36L2) is an RNA-binding protein that destabilizes transcripts containing adenine-uridine rich elements (AREs). The overlap between ZFP36L2 targets in different tissues is minimal, suggesting that ZFP36L2-targeting is highly tissue specific. We developed a novel Zfp36l2-lacking mouse model (L2-fKO) to identify factors governing this tissue specificity. We found 549 upregulated genes in the L2-fKO spleen by RNA-seq. These upregulated genes were enriched in ARE motifs in the 3'UTRs, which suggests that they are ZFP36L2 targets, however the precise sequence requirement for targeting was not evident from motif analysis alone. We therefore used gel-shift mobility assays on 12 novel putative targets and established that ZFP36L2 requires a 7-mer (UAUUUAU) motif to bind. We observed a statistically significant enrichment of 7-mer ARE motifs in upregulated genes and determined that ZFP36L2 targets are enriched for multiple 7-mer motifs. Elavl2 mRNA, which has three 7-mer (UAUUUAU) motifs, was also upregulated in L2-fKO spleens. Overexpression of ZFP36L2, but not a ZFP36L2(C176S) mutant, reduced Elavl2 mRNA expression, suggesting a direct negative effect. Additionally, a reporter assay demonstrated that the ZFP36L2 effect on Elavl2 decay is dependent on the Elavl2-3'UTR and requires the 7-mer AREs. Our data indicate that Elavl2 mRNA is a novel target of ZFP36L2, specific to the spleen. Likely, ZFP36L2 combined with other RNA binding proteins, such as ELAVL2, governs tissue specificity.

Alternative poly-adenylation modulates α1-antitrypsin expression in chronic obstructive pulmonary disease.

α1-anti-trypsin (A1AT), encoded by SERPINA1, is a neutrophil elastase inhibitor that controls the inflammatory response in the lung. Severe A1AT deficiency increases risk for Chronic Obstructive Pulmonary Disease (COPD), however, the role of A1AT in COPD in non-deficient individuals is not well known. We identify a 2.1-fold increase (p = 2.5x10-6) in the use of a distal poly-adenylation site in primary lung tissue RNA-seq in 82 COPD cases when compared to 64 controls and replicate this in an independent study of 376 COPD and 267 controls. This alternative polyadenylation event involves two sites, a proximal and distal site, 61 and 1683 nucleotides downstream of the A1AT stop codon. To characterize this event, we measured the distal ratio in human primary tissue short read RNA-seq data and corroborated our results with long read RNA-seq data. Integrating these results with 3' end RNA-seq and nanoluciferase reporter assay experiments we show that use of the distal site yields mRNA transcripts with over 50-fold decreased translation efficiency and A1AT expression. We identified seven RNA binding proteins using enhanced CrossLinking and ImmunoPrecipitation precipitation (eCLIP) with one or more binding sites in the SERPINA1 3' UTR. We combined these data with measurements of the distal ratio in shRNA knockdown experiments, nuclear and cytoplasmic fractionation, and chemical RNA structure probing. We identify Quaking Homolog (QKI) as a modulator of SERPINA1 mRNA translation and confirm the role of QKI in SERPINA1 translation with luciferase reporter assays. Analysis of single-cell RNA-seq showed differences in the distribution of the SERPINA1 distal ratio among hepatocytes, macrophages, αß-Tcells and plasma cells in the liver. Alveolar Type 1,2, dendritic cells and macrophages also vary in their distal ratio in the lung. Our work reveals a complex post-transcriptional mechanism that regulates alternative polyadenylation and A1AT expression in COPD.

A pipeline for computational design of novel RNA-like topologies.

Designing novel RNA topologies is a challenge, with important therapeutic and industrial applications. We describe a computational pipeline for design of novel RNA topologies based on our coarse-grained RNA-As-Graphs (RAG) framework. RAG represents RNA structures as tree graphs and describes RNA secondary (2D) structure topologies (currently up to 13 vertices, ≈260 nucleotides). We have previously identified novel graph topologies that are RNA-like among these. Here we describe a systematic design pipeline and illustrate design for six broad design problems using recently developed tools for graph-partitioning and fragment assembly (F-RAG). Following partitioning of the target graph, corresponding atomic fragments from our RAG-3D database are combined using F-RAG, and the candidate atomic models are scored using a knowledge-based potential developed for 3D structure prediction. The sequences of the top scoring models are screened further using available tools for 2D structure prediction. The results indicate that our modular approach based on RNA-like topologies rather than specific 2D structures allows for greater flexibility in the design process, and generates a large number of candidate sequences quickly. Experimental structure probing using SHAPE-MaP for two sequences agree with our predictions and suggest that our combined tools yield excellent candidates for further sequence and experimental screening.

An RNA structure-mediated, posttranscriptional model of human α-1-antitrypsin expression.

Chronic obstructive pulmonary disease (COPD) affects over 65 million individuals worldwide, where α-1-antitrypsin deficiency is a major genetic cause of the disease. The α-1-antitrypsin gene, SERPINA1, expresses an exceptional number of mRNA isoforms generated entirely by alternative splicing in the 5'-untranslated region (5'-UTR). Although all SERPINA1 mRNAs encode exactly the same protein, expression levels of the individual mRNAs vary substantially in different human tissues. We hypothesize that these transcripts behave unequally due to a posttranscriptional regulatory program governed by their distinct 5'-UTRs and that this regulation ultimately determines α-1-antitrypsin expression. Using whole-transcript selective 2'-hydroxyl acylation by primer extension (SHAPE) chemical probing, we show that splicing yields distinct local 5'-UTR secondary structures in SERPINA1 transcripts. Splicing in the 5'-UTR also changes the inclusion of long upstream ORFs (uORFs). We demonstrate that disrupting the uORFs results in markedly increased translation efficiencies in luciferase reporter assays. These uORF-dependent changes suggest that α-1-antitrypsin protein expression levels are controlled at the posttranscriptional level. A leaky-scanning model of translation based on Kozak translation initiation sequences alone does not adequately explain our quantitative expression data. However, when we incorporate the experimentally derived RNA structure data, the model accurately predicts translation efficiencies in reporter assays and improves α-1-antitrypsin expression prediction in primary human tissues. Our results reveal that RNA structure governs a complex posttranscriptional regulatory program of α-1-antitrypsin expression. Crucially, these findings describe a mechanism by which genetic alterations in noncoding gene regions may result in α-1-antitrypsin deficiency.

Impact of RNA structure on ZFP36L2 interaction with luteinizing hormone receptor mRNA.

ZFP36L2 (L2) destabilizes AU-rich element (ARE)-containing transcripts and has been implicated in female fertility. We have shown that only one of three putative AREs within the 3' UTR of murine luteinizing hormone receptor mRNA, ARE2197 (UAUUUAU), is capable of interacting with L2. To assess whether structural elements of ARE2197 could explain this unique binding ability, we performed whole-transcript SHAPE-MaP (selective 2' hydroxyl acylation by primer extension-mutational profiling) of the full-length mLHR mRNA. The data revealed that the functional ARE2197 is located in a hairpin loop structure and most nucleotides are highly reactive. In contrast, each of the nonbinding AREs, 2301 and 2444, contains only a pentamer AUUUA; and in ARE2301 much of the ARE sequence is poorly accessible. Because the functional mARE was also found to be conserved in humans at the sequence level (ARE 2223), we decided to investigate whether binding and structure are also preserved. Similar to mouse, only one ARE in hLHR mRNA is capable of binding to L2; and it is also located in a hairpin structure, based on our SHAPE-MaP data. To investigate the role of secondary structure in the binding, we mutated specific nucleotides in both functional AREs. Mutations in the flexible stem region proximal to the loop that enforce strong base-pairing, drastically reduced L2 binding affinity; this confirms that the structural context is critical for L2 recognition of hARE2223. Collectively, our results suggest that a combination of minimal ARE sequence, placement of the ARE in a hairpin loop, and stem flexibility mediate high-affinity L2 binding to hLHR mRNA.

Inducing circular RNA formation using the CRISPR endoribonuclease Csy4.

Circular RNAs (circRNAs) are highly stable, covalently closed RNAs that are regulated in a spatiotemporal manner and whose functions are largely unknown. These molecules have the potential to be incorporated into engineered systems with broad technological implications. Here we describe a switch for inducing back-splicing of an engineered circRNA that relies on the CRISPR endoribonuclease, Csy4, as an activator of circularization. The endoribonuclease activity and 3' end-stabilizing properties of Csy4 are particularly suited for this task. Coexpression of Csy4 and the circRNA switch allows for the removal of downstream competitive splice sites and stabilization of the 5' cleavage product. This subsequently results in back-splicing of the 5' cleavage product into a circRNA that can translate a reporter protein from an internal ribosomal entry site (IRES). Our platform outlines a straightforward approach toward regulating splicing and could find potential applications in synthetic biology as well as in studying the properties of different circRNAs.

Characterization of DeltaN-Zfp36l2 mutant associated with arrest of early embryonic development and female infertility.

The zinc finger protein 36-like 2, Zfp36l2, has been implicated in female mouse infertility, because an amino-terminal truncation mutation (ΔN-Zfp36l2) leads to two-cell stage arrest of embryos derived from the homozygous mutant female gamete. Zfp36l2 is a member of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins that can bind to transcripts containing AU-rich elements (ARE), resulting in deadenylation and destabilization of these transcripts. I show here that the mouse Zfp36l2 is composed of two exons and a single intron, encoding a polypeptide of 484 amino acids. I observed that ΔN-Zfp36l2 protein is similar to both wild-type Zfp36l2 and TTP (Zfp36) in that it shuttles between the cytoplasm and nucleus, binds to RNAs containing AREs, and promotes deadenylation of a model ARE transcript in a cell-based co-transfection assay. Surprisingly, in contrast to TTP, Zfp36l2 mRNA and protein were rapidly down-regulated upon LPS exposure in bone marrow-derived macrophages. The ΔN-Zfp36l2 protein was substantially more resistant to stimulus-induced down-regulation than the WT. I postulate that the embryonic arrest linked to the ΔN-Zfp36l2 truncation might be related to its resistance to stimulus-induced down-regulation.

A single-cell transcriptional gradient in human cutaneous memory T cells restricts Th17/Tc17 identity.

The homeostatic mechanisms that fail to restrain chronic tissue inflammation in diseases, such as psoriasis vulgaris, remain incompletely understood. We profiled transcriptomes and epitopes of single psoriatic and normal skin-resident T cells, revealing a gradated transcriptional program of coordinately regulated inflammation-suppressive genes. This program, which is sharply suppressed in lesional skin, strikingly restricts Th17/Tc17 cytokine and other inflammatory mediators on the single-cell level. CRISPR-based deactivation of two core components of this inflammation-suppressive program, ZFP36L2 and ZFP36, replicates the interleukin-17A (IL-17A), granulocyte macrophage-colony-stimulating factor (GM-CSF), and interferon gamma (IFNγ) elevation in psoriatic memory T cells deficient in these transcripts, functionally validating their influence. Combinatoric expression analysis indicates the suppression of specific inflammatory mediators by individual program members. Finally, we find that therapeutic IL-23 blockade reduces Th17/Tc17 cell frequency in lesional skin but fails to normalize this inflammatory-suppressive program, suggesting how treated lesions may be primed for recurrence after withdrawal of treatment.

RNA-Binding Protein ZFP36L2 Downregulates Helios Expression and Suppresses the Function of Regulatory T Cells.

The zinc finger protein 36-like 2, ZFP36L2, is a member of a small family of RNA-binding proteins composed by ZFP36 (also known as tristetraprolin, TTP), ZFP36L1 and ZFP36L2 in humans, with corresponding murine orthologs. These proteins bind to adenine uridine-rich element (ARE) in the 3'untranslated region of target messenger RNA and stimulate target degradation. ZFP36 functions as an anti-inflammatory modulator in murine models of inflammatory diseases by down-regulating the production of inflammatory cytokines such as tumor necrosis factor-α. However, how ZFP36L1 and ZFP36L2 alter the function of CD4+ T cells is not completely understood. We addressed this issue by searching for the target genes of ZFP36L2 by comprehensive transcriptome analysis. We observed that ZFP36L2 is highly expressed in naïve CD4+ T cells; however, when CD4+ T cells are stimulated through their T cell receptors, ZFP36L2 expression is rapidly reduced in both humans and mice. Among CD4+ T cell populations, the expression levels of ZFP36L2 in regulatory T cells (Tregs) were significantly lower than those in naïve or effector CD4+ T cells. RNA-sequence analysis revealed that the forced expression of ZFP36L2 decreased Ikzf2 (encoding Helios) expression in Foxp3+ Tregs and inhibited the ability of induced Tregs (iTregs). ZFP36L2 directly bound to and destabilized the 3'untranslated region of Ikzf2 mRNA, which contains AU-rich elements. These results indicate that ZFP36L2 reduces the expression of Ikzf2 and suppresses iTreg function, raising the interesting possibility that the inhibition of ZFP36L2 in iTregs could be a therapeutic strategy for autoimmune diseases.

Overexpression of HLA class I molecules on T cells among type 1 diabetes Brazilian patients.

To evaluate the membrane expression of histocompatibility (HLA) class I (A-C) molecules on lymphomononuclear cells involved in the pathogenesis of type 1 diabetes, we studied 20 newly diagnosed Brazilian patients and 20 matched controls. The coexpression of HLA and cluster of differentiation (CD) molecules was evaluated by flow cytometry. Compared to controls, patients presented increased fluorescence intensity of HLA class I molecules on CD3+, CD4+ and CD8+ cells. Patients exhibiting the HLA-DQB1*0201 and/or DQB1*0302 alleles presented increased expression of class I molecules in relation to HLA-matched healthy controls. The increased HLA class I expression in subsets of T cells may be due to the proinflammatory profile of the disease as well as to the presence of diabetes susceptibility alleles.

The potential of the riboSNitch in personalized medicine.

RNA conformation plays a significant role in stability, ligand binding, transcription, and translation. Single nucleotide variants (SNVs) have the potential to disrupt specific structural elements because RNA folds in a sequence-specific manner. A riboSNitch is an element of RNA structure with a specific function that is disrupted by an SNV or a single nucleotide polymorphism (SNP; or polymorphism; SNVs occur with low frequency in the population, <1%). The riboSNitch is analogous to a riboswitch, where binding of a small molecule rather than mutation alters the structure of the RNA to control gene regulation. RiboSNitches are particularly relevant to interpreting the results of genome-wide association studies (GWAS). Often GWAS identify SNPs associated with a phenotype mapping to noncoding regions of the genome. Because a majority of the human genome is transcribed, significant subsets of GWAS SNPs are putative riboSNitches. The extent to which the transcriptome is tolerant of SNP-induced structure change is still poorly understood. Recent advances in ultra high-throughput structure probing begin to reveal the structural complexities of mutation-induced structure change. This review summarizes our current understanding of SNV and SNP-induced structure change in the human transcriptome and discusses the importance of riboSNitch discovery in interpreting GWAS results and massive sequencing projects.

The RNA-binding protein, ZFP36L2, influences ovulation and oocyte maturation.

ZFP36L2 protein destabilizes AU-rich element-containing transcripts and has been implicated in female fertility. In the C57BL/6NTac mouse, a mutation in Zfp36l2 that results in the decreased expression of a form of ZFP36L2 in which the 29 N-terminal amino acid residues have been deleted, ΔN-ZFP36L2, leads to fertilized eggs that arrest at the two-cell stage. Interestingly, homozygous ΔN-Zfp36l2 females in the C57BL/6NTac strain release 40% fewer eggs than the WT littermates (Ramos et al., 2004), suggesting an additional defect in ovulation and/or oocyte maturation. Curiously, the same ΔN-Zfp36l2 mutation into the SV129 strain resulted in anovulation, prompting us to investigate a potential problem in ovulation and oocyte maturation. Remarkably, only 20% of ΔN-Zfp36l2 oocytes in the 129S6/SvEvTac strain matured ex vivo, suggesting a defect on the oocyte meiotic maturation process. Treatment of ΔN-Zfp36l2 oocytes with a PKA inhibitor partially rescued the meiotic arrested oocytes. Furthermore, cAMP levels were increased in ΔN-Zfp36l2 oocytes, linking the cAMP/PKA pathway and ΔN-Zfp36l2 with meiotic arrest. Since ovulation and oocyte maturation are both triggered by LHR signaling, the downstream pathway was investigated. Adenylyl cyclase activity was increased in ΔN-Zfp36l2 ovaries only upon LH stimulation. Moreover, we discovered that ZFP36L2 interacts with the 3'UTR of LHR mRNA and that decreased expression levels of Zfp36l2 correlates with higher levels of LHR mRNA in synchronized ovaries. Furthermore, overexpression of ZFP36L2 decreases the endogenous expression of LHR mRNA in a cell line. Therefore, we propose that lack of the physiological down regulation of LHR mRNA levels by ZFP36L2 in the ovaries is associated with anovulation and oocyte meiotic arrest.

A unique C-terminal repeat domain maintains the cytosolic localization of the placenta-specific tristetraprolin family member ZFP36L3.

Members of the tristetraprolin family of CCCH tandem zinc finger proteins bind to AU-rich elements in certain cellular mRNAs, leading to their deadenylation and destabilization. Studies in knock-out mice demonstrated roles for three of the family members, tristetraprolin, ZFP36L1, and ZFP36L2, in inflammation, chorioallantoic fusion, and early embryonic development, respectively. However, little is known about a recently discovered placenta-specific tristetraprolin family member, ZFP36L3. Tristetraprolin, ZFP36L1, and ZFP36L2 have been shown to shuttle between the nucleus and cytoplasm, using typical hydrophobic amino acid-rich nuclear export sequences, and nuclear localization sequences located within the tandem zinc finger domain. In contrast, we previously showed that green fluorescent protein-labeled ZFP36L3, expressed in HEK 293 cells, remained cytosolic, even in the presence of the nuclear export blocker leptomycin B. We show here that the conserved tandem zinc finger domain contains an active nuclear localization signal. However, the sequence corresponding to the nuclear export signal in the other family members was nonfunctional, and thus did not contribute to the cytosolic localization. The unique C-terminal repeat domain could override the activity of the nuclear localization sequence, preventing the import of ZFP36L3 into the nucleus. Immunostaining of mouse placenta demonstrated that ZFP36L3 was located only in the cytoplasm of trophoblast cells. Thus, in contrast to the other mammalian members of this protein family, ZFP36L3 is a "full-time" cytosolic protein, rather than a nucleocytoplasmic shuttling protein. The significance of this difference in subcellular localization to the physiology of placental trophoblast cells, where ZFP36L3 is selectively expressed, remains to be determined.

Zfp36l3, a rodent X chromosome gene encoding a placenta-specific member of the Tristetraprolin family of CCCH tandem zinc finger proteins.

Members of the tristetraprolin (TTP) family of CCCH tandem zinc finger (TZF) proteins can bind directly to AU-rich elements (ARE) in mRNA, causing deadenylation and destabilization of the transcripts to which they bind. We describe here a novel fourth mammalian member of the TTP protein family, designated ZFP36L3, which could also bind directly to ARE-containing RNAs and could promote the deadenylation and degradation of ARE-containing target RNAs. Zfp36l3 transcript expression was detected only in placenta and extraembryonic tissues in the mouse. It was expressed throughout development in the placenta and was particularly highly expressed in the cells of the labyrinthine layer of the trophoblastic placenta. Unlike the other family members, the expression of a ZFP36L3-green fluorescent protein fusion protein was entirely cytoplasmic when expressed in 293 cells, even in the presence of the CRM1-dependent nuclear export inhibitor leptomycin B. Zfp36l3 was located on the mouse X chromosome; a similar predicted gene was present on the rat X chromosome, but there was no evidence for a similar gene in humans. ZFP36L3 may thus be a rodent-specific or even murine-specific member of the TTP protein family. Its presumed role in placental physiology may be unique to rodents or murine rodents, but this role may be subsumed by other family members in nonrodents.

Members of the tristetraprolin family of tandem CCCH zinc finger proteins exhibit CRM1-dependent nucleocytoplasmic shuttling.

Members of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins can bind directly to certain types of AU-rich elements (AREs) in mRNA. Experiments in TTP-deficient mice have shown that TTP is involved in the physiological destabilization of at least two cytokine mRNAs, those encoding tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor. The two other known mammalian members of the TTP family, CMG1 and TIS11D, also contain ARE-binding CCCH tandem zinc finger domains and can also destabilize ARE-containing mRNAs. To investigate the effects of primary sequence on the subcellular localization of these proteins, we constructed green fluorescent protein fusions with TTP, CMG1, and TIS11D; these were predominantly cytoplasmic when expressed in 293 or HeLa cells. Deletion and mutation analyses revealed functional nuclear export signals in the amino terminus of TTP and in the carboxyl termini of CMG1 and TIS11D. This type of leucine-rich nuclear export signal interacts with the nuclear export receptor CRM1; abrogation of CRM1 activity resulted in nuclear accumulation of TTP, CMG1, and TIS11D. These proteins are thus nucleocytoplasmic shuttling proteins and rely on CRM1 for their export from the nucleus. Although TTP, CMG1, and TIS11D lack known nuclear import sequences, mapping experiments revealed that their nuclear accumulation required an intact tandem zinc finger domain but did not require RNA binding ability. These findings suggest possible roles for nuclear import and export in the regulation of cellular TTP, CMG1, and TIS11D activity.

The CCCH tandem zinc-finger protein Zfp36l2 is crucial for female fertility and early embryonic development.

The CCCH tandem zinc finger protein, Zfp36l2, like its better-known relative tristetraprolin (TTP), can decrease the stability of AU-rich element-containing transcripts in cell transfection studies; however, its physiological importance is unknown. We disrupted Zfp36l2 in mice, resulting in decreased expression of a truncated protein in which the N-terminal 29 amino acids had been deleted (DeltaN-Zfp36l2). Mice derived from different clones of ES cells exhibited complete female infertility, despite evidence from embryo and ovary transplantation experiments that they could gestate and rear wild-type young. DeltaN-Zfp36l2 females apparently cycled and ovulated normally, and their ova could be fertilized; however, the embryos did not progress beyond the two-cell stage of development. These mice represent a specific model of disruption of the earliest stages of embryogenesis, implicating Zfp36l2, a probable mRNA-binding and destabilizing protein, in the physiological control of female fertility at the level of early embryonic development. This newly identified biological role for Zfp36l2 may have implications for maternal mRNA turnover in normal embryogenesis, and conceivably could be involved in some cases of unexplained human female infertility.