Posttranscriptional RNA Metabolism Research Articles

Insulin-like growth factor-2 mRNA-binding protein 2 facilitates post-ischemic angiogenesis by increasing the stability of fibroblast growth factor 2 mRNA and its protein expression

BackgroundPost-ischemic angiogenesis is crucial for reestablishing blood flow in conditions such as peripheral artery disease (PAD). The role of insulin-like growth factor-2 mRNA-binding protein 2 (IGF2BP2) in post-transcriptional RNA metabolism and its involvement in post-ischemic angiogenesis remains unclear. MethodsUsing a human GEO database and a hind-limb ischemia (HLI) mouse model, the predominant isoform IGF2BP2 in ischemic gastrocnemius tissue was identified. Adeno-associated virus with the Tie1 promoter induced IGF2BP2 overexpression in the HLI model, evaluating the expression of vascular structural proteins (CD31 and α-SMA) and blood flow recovery after HLI. In vitro experiments with human umbilical vein endothelial cells (HUVECs) demonstrated that lentivirus-mediated IGF2BP2 overexpression upregulates cell proliferation, migration, and tube formation. GeneCards, RNAct databases, and subsequent reverse transcription quantitative polymerase chain reaction (RT-qPCR) predicted IGF2BP2 interactions with fibroblast growth factor 2 (FGF2) mRNA, and actinomycin D treatment, binding site predictions and CLIP-seq data further confirmed this interaction. Furthermore, western blotting, enzyme-linked immunosorbent assay, and RNA immunoprecipitation followed by RT-qPCR were performed to validate IGF2BP2's interaction with FGF2 mRNA and to assess its role in stabilizing FGF2 mRNA, as well as its impact on FGF2 protein expression. ResultsHLI reduced IGF2BP2 expression in the gastrocnemius tissue, which gradually increased during blood flow recovery. IGF2BP2 overexpression in HLI mice accelerated blood flow recovery and increased capillary and small artery densities. The overexpression of IGF2BP2 in HUVECs stimulated proliferation, migration, and tube formation by interacting with FGF2 mRNA to increase its stability. This interaction resulted in increased levels of FGF2 protein and secretion, ultimately promoting angiogenesis. ConclusionsIGF2BP2 contributes to blood flow restoration post-ischemia in vivo and promotes angiogenesis in HUVECs by enhancing FGF2 mRNA stability and FGF2 protein expression and secretion. These findings underscore IGF2BP2's therapeutic potential in ischemic conditions, such as PAD.

Liver cancer initiation requires translational activation by an oncofetal regulon involving LIN28 proteins.

It is unknown which posttranscriptional regulatory mechanisms are required for oncogenic competence. Here, we show that the LIN28 family of RNA-binding proteins (RBPs), which facilitate posttranscriptional RNA metabolism within ribonucleoprotein networks, is essential for the initiation of diverse oncotypes of hepatocellular carcinoma (HCC). In HCC models driven by NRASG12V/Tp53, CTNNB1/YAP/Tp53, or AKT/Tp53, mice without Lin28a and Lin28b were markedly impaired in cancer initiation. We biochemically defined an oncofetal regulon of 15 factors connected to LIN28 through direct mRNA and protein interactions. Interestingly, all were RBPs and only 1 of 15 was a Let-7 target. Polysome profiling and reporter assays showed that LIN28B directly increased the translation of 8 of these 15 RBPs. As expected, overexpression of LIN28B and IGFBP1-3 was able to genetically rescue cancer initiation. Using this platform to probe components downstream of LIN28, we found that 8 target RBPs were able to restore NRASG12V/Tp53 cancer formation in Lin28a/Lin28b-deficient mice. Furthermore, these LIN28B targets promote cancer initiation through an increase in protein synthesis. LIN28B, central to an RNP regulon that increases translation of RBPs, is important for tumor initiation in the liver.

An update on post-transcriptional regulation of retrotransposons.

Retrotransposons, including LINE-1, Alu, SVA, and endogenous retroviruses, are one of the major constituents of human genomic repetitive sequences. Through the process of retrotransposition, some of them occasionally insert into new genomic locations by a copy-paste mechanism involving RNA intermediates. Irrespective of de novo genomic insertions, retrotransposon expression can lead to DNA double-strand breaks and stimulate cellular innate immunity through endogenous patterns. As a result, retrotransposons are tightly regulated by multi-layered regulatory processes to prevent the dangerous effects of their expression. In recent years, significant progress was made in revealing how retrotransposon biology intertwines with general post-transcriptional RNA metabolism. Here, I summarize current knowledge on the involvement of post-transcriptional factors in the biology of retrotransposons, focusing on LINE-1. I emphasize general RNA metabolisms such as methylation of adenine (m6 A), RNA 3'-end polyadenylation and uridylation, RNA decay and translation regulation. I discuss the effects of retrotransposon RNP sequestration in cytoplasmic bodies and autophagy. Finally, I summarize how innate immunity restricts retrotransposons and how retrotransposons make use of cellular enzymes, including the DNA repair machinery, to complete their replication cycles.

Phase separation of HRLP regulates flowering time in Arabidopsis.

RNA binding proteins mediate posttranscriptional RNA metabolism and play regulatory roles in many developmental processes in eukaryotes. Despite their known effects on the floral transition from vegetative to reproductive growth in plants, the underlying mechanisms remain largely obscure. Here, we show that a hitherto unknown RNA binding protein, hnRNP R-LIKE PROTEIN (HRLP), inhibits cotranscriptional splicing of a key floral repressor gene FLOWERING LOCUS C (FLC). This, in turn, facilitates R-loop formation near FLC intron I to repress its transcription, thereby promoting the floral transition in Arabidopsis thaliana. HRLP, together with the splicing factor ARGININE/SERINE-RICH 45, forms phase-separated nuclear condensates with liquid-like properties, which is essential for HRLP function in regulating FLC splicing, R-loop formation, and RNA Polymerase II recruitment. Our findings reveal that inhibition of cotranscriptional splicing of FLC by nuclear HRLP condensates constitutes the molecular basis for down-regulation of FLC transcript levels to ensure the reproductive success of Arabidopsis.

Inactivation of RNase P in Escherichia coli significantly changes post-transcriptional RNA metabolism.

Ribonuclease P (RNase P), which is required for the 5'-end maturation of tRNAs in every organism, has been shown to play a limited role in other aspects of RNA metabolism in Escherichia coli. Using RNA-sequencing (RNA-seq), we demonstrate that RNase P inactivation affects the abundances of ~46% of the expressed transcripts in E. coli and provide evidence that its essential function is its ability to generate pre-tRNAs from polycistronic tRNA transcripts. The RNA-seq results agreed with the published data and northern blot analyses of 75/83 transcripts (mRNAs, sRNAs, and tRNAs). Changes in transcript abundances in the RNase P mutant also correlated with changes in their half-lives. Inactivating the stringent response did not alter the rnpA49 phenotype. Most notably, increases in the transcript abundances were observed for all genes in the cysteine regulons, multiple toxin-antitoxin modules, and sigma S-controlled genes. Surprisingly, poly(A) polymerase (PAP I) modulated the abundances of ~10% of the transcripts affected by RNase P. A comparison of the transcriptomes of RNase P, RNase E, and RNase III mutants suggests that they affect distinct substrates. Together, our work strongly indicates that RNase P is a major player in all aspects of post-transcriptional RNA metabolism in E. coli.

Roles of Organellar RNA-Binding Proteins in Plant Growth, Development, and Abiotic Stress Responses.

Organellar gene expression (OGE) in chloroplasts and mitochondria is primarily modulated at post-transcriptional levels, including RNA processing, intron splicing, RNA stability, editing, and translational control. Nucleus-encoded Chloroplast or Mitochondrial RNA-Binding Proteins (nCMRBPs) are key regulatory factors that are crucial for the fine-tuned regulation of post-transcriptional RNA metabolism in organelles. Although the functional roles of nCMRBPs have been studied in plants, their cellular and physiological functions remain largely unknown. Nevertheless, existing studies that have characterized the functions of nCMRBP families, such as chloroplast ribosome maturation and splicing domain (CRM) proteins, pentatricopeptide repeat (PPR) proteins, DEAD-Box RNA helicase (DBRH) proteins, and S1-domain containing proteins (SDPs), have begun to shed light on the role of nCMRBPs in plant growth, development, and stress responses. Here, we review the latest research developments regarding the functional roles of organellar RBPs in RNA metabolism during growth, development, and abiotic stress responses in plants.

Analysis of post-transcriptional RNA metabolism in prokaryotes.

Post-transcriptional RNA metabolic pathways play important roles in permitting prokaryotes to operate under a variety of environmental conditions. Although significant progress has been made during the last decade in deciphering RNA processing pathways in a number of bacteria, a complete understanding of post-transcriptional RNA metabolism in any single microorganism is far from reality. Here we describe multiple experimental approaches that can be used to study mRNA stability, tRNA and rRNA processing, sRNA metabolism, and polyadenylation in prokaryotes. The methods described here can be readily utilized in both Gram-negative and Gram-positive bacteria with simple modifications.

Enzymes Involved in Posttranscriptional RNA Metabolism in Gram-Negative Bacteria.

Gene expression in Gram-negative bacteria is regulated at many levels, including transcription initiation, RNA processing, RNA/RNA interactions, mRNA decay, and translational controls involving enzymes that alter translational efficiency. In this review, we discuss the various enzymes that control transcription, translation, and RNA stability through RNA processing and degradation. RNA processing is essential to generate functional RNAs, while degradation helps control the steady-state level of each individual transcript. For example, all the pre-tRNAs are transcribed with extra nucleotides at both their 5' and 3' termini, which are subsequently processed to produce mature tRNAs that can be aminoacylated. Similarly, rRNAs that are transcribed as part of a 30S polycistronic transcript are matured to individual 16S, 23S, and 5S rRNAs. Decay of mRNAs plays a key role in gene regulation through controlling the steady-state level of each transcript, which is essential for maintaining appropriate protein levels. In addition, degradation of both translated and nontranslated RNAs recycles nucleotides to facilitate new RNA synthesis. To carry out all these reactions, Gram-negative bacteria employ a large number of endonucleases, exonucleases, RNA helicases, and poly(A) polymerase, as well as proteins that regulate the catalytic activity of particular RNases. Under certain stress conditions, an additional group of specialized endonucleases facilitate the cell's ability to adapt and survive. Many of the enzymes, such as RNase E, RNase III, polynucleotide phosphorylase, RNase R, and poly(A) polymerase I, participate in multiple RNA processing and decay pathways.

Genome-Wide Maps of m6A circRNAs Identify Widespread and Cell-Type-Specific Methylation Patterns that Are Distinct from mRNAs

N6-methyladenosine (m6A) is the most abundant internal modification of mRNAs and is implicated in all aspects of post-transcriptional RNA metabolism. However, little is known about m6A modifications to circular (circ) RNAs. We developed a computational pipeline (AutoCirc) that, together with depletion of ribosomal RNA and m6A immunoprecipitation, defined thousands of m6A circRNAs with cell-type-specific expression. The presence of m6A circRNAs is corroborated by interaction between circRNAs and YTHDF1/YTHDF2, proteins that read m6A sites in mRNAs, and by reduced m6A levels upon depletion of METTL3, the m6A writer. Despite sharing m6A readers and writers, m6A circRNAs are frequently derived from exons that are not methylated in mRNAs, whereas mRNAs that are methylated on the same exons that compose m6A circRNAs exhibit less stability in aprocess regulated by YTHDF2. These results expand our understanding of the breadth of m6A modifications and uncover regulation of circRNAs through m6A modification.

Antisense transcription licenses nascent transcripts to mediate transcriptional gene silencing.

In eukaryotes, antisense transcription can regulate sense transcription by induction of epigenetic modifications. We showed previously that antisense transcription triggers Dicer-independent siRNA (disiRNA) production and disiRNA locus DNA methylation (DLDM) in Neurospora crassa Here we show that the conserved exonuclease ERI-1 (enhanced RNAi-1) is a critical component in this process. Antisense transcription and ERI-1 binding to target RNAs are necessary and sufficient to trigger DLDM. Convergent transcription causes stalling of RNA polymerase II during transcription, which permits ERI-1 to bind nascent RNAs in the nucleus and recruit a histone methyltransferase complex that catalyzes chromatin modifications. Furthermore, we show that, in the cytoplasm, ERI-1 targets hundreds of transcripts from loci without antisense transcription to regulate RNA stability. Together, our results demonstrate a critical role for transcription kinetics in long noncoding RNA-mediated epigenetic modifications and identify ERI-1 as an important regulator of cotranscriptional gene silencing and post-transcriptional RNA metabolism.

Emerging Roles of RNA-Binding Proteins in Plant Growth, Development, and Stress Responses.

Posttranscriptional regulation of RNA metabolism, including RNA processing, intron splicing, editing, RNA export, and decay, is increasingly regarded as an essential step for fine-tuning the regulation of gene expression in eukaryotes. RNA-binding proteins (RBPs) are central regulatory factors controlling posttranscriptional RNA metabolism during plant growth, development, and stress responses. Although functional roles of diverse RBPs in living organisms have been determined during the last decades, our understanding of the functional roles of RBPs in plants is lagging far behind our understanding of those in other organisms, including animals, bacteria, and viruses. However, recent functional analysis of multiple RBP family members involved in plant RNA metabolism and elucidation of the mechanistic roles of RBPs shed light on the cellular roles of diverse RBPs in growth, development, and stress responses of plants. In this review, we will discuss recent studies demonstrating the emerging roles of multiple RBP family members that play essential roles in RNA metabolism during plant growth, development, and stress responses.

A nuclear-encoded chloroplast-targeted S1 RNA-binding domain protein affects chloroplast rRNA processing and is crucial for the normal growth of Arabidopsis thaliana.

Despite the fact that a variety of nuclear-encoded RNA-binding proteins (RBPs) are targeted to the chloroplast and play essential roles during post-transcriptional RNA metabolism in the chloroplast, the physiological roles of the majority of chloroplast-targeted RBPs remain elusive. Here, we investigated the functional role of a nuclear-encoded S1 domain-containing RBP, designated SDP, in the growth and development of Arabidopsis thaliana. Confocal analysis of the SDP-green fluorescent protein revealed that SDP was localized to the chloroplast. The loss-of-function sdp mutant displayed retarded seed germination and pale-green phenotypes, and grew smaller than the wild-type plants. Chlorophyll a content and photosynthetic activity of the sdp mutant were much lower than those of wild-type plants, and the structures of the chloroplast and the prolamellar body were abnormal in the sdp mutant. The processing of rRNAs in the chloroplast was defective in the sdp mutant, and SDP was able to bind chloroplast 23S, 16S, 5S and 4.5S rRNAs. Notably, SDP possesses RNA chaperone activity. Transcript levels of the nuclear genes involved in chlorophyll biosynthesis were altered in the sdp mutant. Collectively, these results suggest that chloroplast-targeted SDP harboring RNA chaperone activity affects rRNA processing, chloroplast biogenesis and photosynthetic activity, which is crucial for normal growth of Arabidopsis.

Nuclear Fragile X Mental Retardation Protein Is localized to Cajal Bodies

Fragile X syndrome is caused by loss of function of a single gene encoding the Fragile X Mental Retardation Protein (FMRP). This RNA-binding protein, widely expressed in mammalian tissues, is particularly abundant in neurons and is a component of messenger ribonucleoprotein (mRNP) complexes present within the translational apparatus. The absence of FMRP in neurons is believed to cause translation dysregulation and defects in mRNA transport essential for local protein synthesis and for synaptic development and maturation. A prevalent model posits that FMRP is a nucleocytoplasmic shuttling protein that transports its mRNA targets from the nucleus to the translation machinery. However, it is not known which of the multiple FMRP isoforms, resulting from the numerous alternatively spliced FMR1 transcripts variants, would be involved in such a process. Using a new generation of anti-FMRP antibodies and recombinant expression, we show here that the most commonly expressed human FMRP isoforms (ISO1 and 7) do not localize to the nucleus. Instead, specific FMRP isoforms 6 and 12 (ISO6 and 12), containing a novel C-terminal domain, were the only isoforms that localized to the nuclei in cultured human cells. These isoforms localized to specific p80-coilin and SMN positive structures that were identified as Cajal bodies. The Cajal body localization signal was confined to a 17 amino acid stretch in the C-terminus of human ISO6 and is lacking in a mouse Iso6 variant. As FMRP is an RNA-binding protein, its presence in Cajal bodies suggests additional functions in nuclear post-transcriptional RNA metabolism. Supporting this hypothesis, a missense mutation (I304N), known to alter the KH2-mediated RNA binding properties of FMRP, abolishes the localization of human FMRP ISO6 to Cajal bodies. These findings open unexplored avenues in search for new insights into the pathophysiology of Fragile X Syndrome.

An ABA-regulated putative RNA-binding protein affects seed germination of Arabidopsis under ABA or abiotic stress conditions

Diverse RNA-binding proteins (RBPs) have increasingly been determined to perform crucial roles in posttranscriptional regulation of RNA metabolism during plant response to abscisic acid (ABA) or abiotic stresses, but the functional roles of a variety of ABA-responsive RBPs in ABA and stress response remain poorly understood. Here, we characterized an ABA-regulated putative RBP (At3g54770) in Arabidopsis thaliana, designated ARP1, for its role in response to ABA or abiotic stresses. The expression of ARP1 was markedly downregulated by ABA, and ARP1 is localized to the nucleus. Interestingly, both overexpression and knockout of ARP1 resulted in delayed seed germination under ABA, high salt, or dehydration stress conditions. The identical phenotypes of ARP1-overexpressing plants and the arp1 null mutant indicate that a tight regulation of ARP1 transcript is required for normal function. Transcript levels of several germination-responsive genes were modulated by ARP1 under ABA. Taken together, the results of the present study demonstrate that ARP1 affects ABA-regulated seed germination of Arabidopsis, and indicate that the regulation of posttranscriptional RNA metabolism is critical in plant response to ABA or abiotic stresses.