Massively parallel analysis of human 3′ UTRs reveals that AU-rich element length and registration predict mRNA destabilization

AU-rich elements (AREs) are 3′ UTR cis-regulatory elements that regulate the stability of mRNAs. Consensus ARE motifs have been determined, but little is known about how differences in 3′ UTR sequences that conform to these motifs affect their function. Here we use functional annotation of sequences from 3′ UTRs (fast-UTR), a massively parallel reporter assay (MPRA), to investigate the effects of 41,288 3′ UTR sequence fragments from 4,653 transcripts on gene expression and mRNA stability in Jurkat and Beas2B cells. Our analyses demonstrate that the length of an ARE and its registration (the first and last nucleotides of the repeating ARE motif) have significant effects on gene expression and stability. Based on this finding, we propose improved ARE classification and concomitant methods to categorize and predict the effect of AREs on gene expression and stability. Finally, to investigate the advantages of our general experimental design we examine other motifs including constitutive decay elements (CDEs), where we show that the length of the CDE stem-loop has a significant impact on steady-state expression and mRNA stability. We conclude that fast-UTR, in conjunction with our analytical approach, can produce improved yet simple sequence-based rules for predicting the activity of human 3′ UTRs.

Conceptual Advances in Control of Inflammation by the RNA-Binding Protein Tristetraprolin

Regulated changes in mRNA stability are critical drivers of gene expression adaptations to immunological cues. mRNA stability is controlled mainly by RNA-binding proteins (RBPs) which can directly cleave mRNA but more often act as adaptors for the recruitment of the RNA-degradation machinery. One of the most prominent RBPs with regulatory roles in the immune system is tristetraprolin (TTP). TTP targets mainly inflammation-associated mRNAs for degradation and is indispensable for the resolution of inflammation as well as the maintenance of immune homeostasis. Recent advances in the transcriptome-wide knowledge of mRNA expression and decay rates together with TTP binding sites in the target mRNAs revealed important limitations in our understanding of molecular mechanisms of TTP action. Such orthogonal analyses lead to the discovery that TTP binding destabilizes some bound mRNAs but not others in the same cell. Moreover, comparisons of various immune cells indicated that an mRNA can be destabilized by TTP in one cell type while it remains stable in a different cell linage despite the presence of TTP. The action of TTP extends from mRNA destabilization to inhibition of translation in a subset of targets. This article will discuss these unexpected context-dependent functions and their implications for the regulation of immune responses. Attention will be also payed to new insights into the role of TTP in physiology and tissue homeostasis.

MicroRNAs in Woody Plants

MicroRNAs (miRNAs) are small (∼21-nucleotides) non-coding RNAs found in plant and animals. MiRNAs function as critical post-transcriptional regulators of gene expression by binding to complementary sequences in their target mRNAs, leading to mRNA destabilization and translational inhibition. Plant miRNAs have some distinct characteristics compared to their animal counterparts, including greater evolutionary conservation and unique miRNA processing methods. The lifecycle of a plant begins with embryogenesis and progresses through seed germination, vegetative growth, reproductive growth, flowering and fruiting, and finally senescence and death. MiRNAs participate in the transformation of plant growth and development and directly monitor progression of these processes and the expression of certain morphological characteristics by regulating transcription factor genes involved in cell growth and differentiation. In woody plants, a large and rapidly increasing number of miRNAs have been identified, but their biological functions are largely unknown. In this review, we summarize the progress of miRNA research in woody plants to date. In particular, we discuss the potential roles of these miRNAs in growth, development, and biotic and abiotic stresses responses in woody plants.

P23 Acts as Functional RBP in the Macrophage Inflammation Response

Macrophages exert the primary cellular immune response. Pathogen components like bacterial lipopolysaccharides (LPS) stimulate macrophage migration, phagocytotic activity and cytokine expression. Previously, we identified the poly(A)+ RNA interactome of RAW 264.7 macrophages. Of the 402 RNA-binding proteins (RBPs), 32 were classified as unique in macrophages, including nineteen not reported to interact with nucleic acids before. Remarkably, P23 a HSP90 co-chaperone, also known as cytosolic prostaglandin E2 synthase (PTGES3), exhibited differential poly(A)+ RNA binding in untreated and LPS-induced macrophages. To identify mRNAs bound by P23 and to elucidate potential regulatory RBP functions in macrophages, we immunoprecipitated P23 from cytoplasmic extracts of cross-linked untreated and LPS-induced cells. RNAseq revealed that enrichment of 44 mRNAs was reduced in response to LPS. Kif15 mRNA, which encodes kinesin family member 15 (KIF15), a motor protein implicated in cytoskeletal reorganization and cell mobility was selected for further analysis. Noteworthy, phagocytic activity of LPS-induced macrophages was enhanced by P23 depletion. Specifically, in untreated RAW 264.7 macrophages, decreased P23 results in Kif15 mRNA destabilization, diminished KIF15 expression and accelerated macrophage migration. We show that the unexpected RBP function of P23 contributes to the regulation of macrophage phagocytotic activity and migration.

The functional characterization of phosphorylation of tristetraprolin at C-terminal NOT1-binding domain

Background Tristetraprolin (TTP) family proteins contain conserved tandem CCCH zinc-finger binding to AU-rich elements and C-terminal NOT1-binding domain. TTP is phosphorylated extensively in cells, and its mRNA destabilization activity is regulated by protein phosphorylation. Methods We generated an antibody against phospho-Serine316 located at the C-terminal NOT1-binding site and examined TTP phosphorylation in LPS-stimulated RAW264.7 cells. Knockout of TTP was created in RAW264.7 cells using CRISPR/Cas9 gene editing to explore TTP functions. Results We demonstrated that Ser316 was phosphorylated by p90 ribosomal S6 kinase 1 (RSK1) and p38-activated protein kinase (MK2) and dephosphorylated by Protein Phosphatase 2A (PP2A). A phosphorylation-mimic mutant of S316D resulted in dissociation with the CCR4-NOT deadenylase complex through weakening interaction with CNOT1. Furthermore, Ser316 and serines 52 and 178 were independently contributed to the CCR4-NOT complex recruitment in the immunoprecipitation assay using phosphor-mimic mutants. In RAW264.7 macrophages, TTP was induced, and Ser316 was phosphorylated through RSK1 and MK2 by LPS stimulation. Knockout of TTP resulted in TNFα mRNA increased due to mRNA stabilization. Overexpression of non-phosphorylated S316A TTP mutant can restore TTP activity and lead to TNFα mRNA decreased. GST pull-down and RNA pull-down analyses demonstrated that endogenous TTP with Ser316 phosphorylation decreased the interaction with CNOT1. Conclusions Our results suggest that the TTP-mediated mRNA stability is modulated by Ser316 phosphorylation via regulating the TTP interaction with the CCR4-NOT deadenylase complex.

Mitotic checkpoint gene expression is tuned by coding sequences

The mitotic checkpoint (also called spindle assembly checkpoint, SAC) is a signaling pathway that safeguards proper chromosome segregation. Proper functioning of the SAC depends on adequate protein concentrations and appropriate stoichiometries between SAC proteins. Yet very little is known about SAC gene expression. Here, we show in fission yeast (S. pombe) that a combination of short mRNA half-lives and long protein half-lives supports stable SAC protein levels. For the SAC genes mad2+ and mad3+, their short mRNA half-lives depend on a high frequency of non-optimal codons and mRNA destabilization mediated through the RNA helicase Ste13 (S.c. Dhh1). In contrast, mad1+ mRNA half-life is short despite a relatively high frequency of optimal codons and despite the lack of known destabilizing motifs in its mRNA. Hence, although they are functionally related, different SAC genes employ different strategies of expression. Taken together, we propose that the codon usage of SAC genes is fine-tuned for proper SAC function. Our work shines light on the gene expression features that promote spindle assembly checkpoint function and suggests that synonymous mutations may weaken the checkpoint.

Investigation of RNA metabolism through large-scale genetic interaction profiling in yeast

Gene deletion and gene expression alteration can lead to growth defects that are amplified or reduced when a second mutation is present in the same cells. We performed 154 genetic interaction mapping (GIM) screens with mutants related with RNA metabolism and measured growth rates of about 700 000 Saccharomyces cerevisiae double mutant strains. The screens used the gene deletion collection in addition to a set of 900 strains in which essential genes were affected by mRNA destabilization (DAmP). To analyze the results we developed RECAP, a strategy that validates genetic interaction profiles by comparison with gene co-citation frequency, and identified links between 1 471 genes and 117 biological processes. To validate specific results, we tested and confirmed a link between an inositol polyphosphate hydrolase complex and mRNA translation initiation. Altogether, the results and the newly developed analysis strategy should represent a useful resource for discovery of gene function in yeast.

The Functional Characterization of Phosphorylation of Tristetraprolin at C-Terminal NOT1-binding Domain

Backgound: Tristetraprolin (TTP) family proteins contain conserved tandem CCCH zinc-finger binding to AU-rich elements and C-terminal NOT1-binding domain. TTP is phosphorylated extensively in cells and its mRNA destabilization activity is regulated by protein phosphorylation. Methods: We generated an antibody against phospho-Serine 316 located at C-terminal NOT1-binding site and examined TTP phosphorylation in LPS-stimulated RAW264.7 cells. Knockout of TTP in RAW264.7 cells using CRISPR/Cas9 gene editing was created to explore TTP functions. Results: We demonstrated that Ser316 was phosphorylated by p90 ribosomal S6 kinase 1 (RSK1) and p38-activated protein kinase (MK2), and dephosphorylated by Protein Phosphatase 2A (PP2A). Phosphorylation-mimic mutant of S316D resulted in dissociation with CCR4-NOT deadenylase complex through weakening interaction with CNOT1. Furthermore, Ser316 and serines 52 and 178 were independently contributed to CCR4-NOT complex recruitment in the immunoprecipitation assay using phosphor-mimic mutants. In RAW264.7 macrophages, TTP was induced and Ser316 was phosphorylated through RSK1 and MK2 by LPS stimulation. Knockout of TTP resulted in TNFα mRNA increased due to mRNA stabilization. Overexpression of non-phosphorylated S316A TTP mutant can restore TTP activity and lead to TNFα mRNA decreased. GST pull-down and RNA pull-down analyses demonstrated that endogenous TTP with Ser316 phosphorylation decreased the interaction with CNOT1. Conclusions: Our results suggest that the TTP-mediated mRNA stability is modulated by Ser316 phosphorylation to regulate the TTP interaction with CCR4-NOT deadenylase complex.