Noncanonical function of DGCR8 controls mESC exit from pluripotency

Mouse embryonic stem cells (mESCs) deficient for DGCR8, a key component of the microprocessor complex, present strong differentiation defects. However, the exact reasons impairing their commitment remain elusive. The analysis of newly generated mutant mESCs revealed that DGCR8 is essential for the exit from the pluripotency state. To dissociate canonical versus noncanonical functions of DGCR8, we complemented the mutant mESCs with a phosphomutant DGCR8, which restored microRNA levels but did not rescue the exit from pluripotency defect. Integration of omics data and RNA immunoprecipitation experiments established DGCR8 as a direct interactor of Tcf7l1 mRNA, a core component of the pluripotency network. Finally, we found that DGCR8 facilitated the splicing of Tcf7l1, an event necessary for the differentiation of mESCs. Our data reveal a new noncanonical function of DGCR8 in the modulation of the alternative splicing of Tcf7l1 mRNA in addition to its established function in microRNA biogenesis

The 5' untranslated region of the serotonin receptor 2C pre-mRNA generates miRNAs and is expressed in non-neuronal cells

The serotonin receptor 2C (HTR2C) gene encodes a G protein-coupled receptor that is exclusively expressed in neurons. Here, we report that the 5' untranslated region of the receptor pre-mRNA as well as its hosted miRNAs is widely expressed in non-neuronal cell lines. Alternative splicing of HTR2C is regulated by MBII-52. MBII-52 and the neighboring MBII-85 cluster are absent in people with Prader-Willi syndrome, which likely causes the disease. We show that MBII-52 and MBII-85 increase expression of the HTR2C 5' UTR and influence expression of the hosted miRNAs. The data indicate that the transcriptional unit expressing HTR2C is more complex than previously recognized and likely deregulated in Prader-Willi syndrome.This work was supported by NIH RO1 GM083187, P20RR020171 to SS; GM079549 to RS and JS; Binational Science Foundation (BSF), USA-Israel, transformative Grant, #2010508, to SS and RS. EE and AP were supported by the Spanish Ministry of Science with grant BIO2011-23920 and by Sandra Ibarra Foundation for Cancer with grant FSI 2011-03

Dual Function of C/D Box Small Nucleolar RNAs in rRNA Modification and Alternative Pre-mRNA Splicing

C/D box small nucleolar RNAs (SNORDs) are small noncoding RNAs, and their best-understood function is to target the methyltransferase fibrillarin to rRNA (for example, SNORD27 performs 2′-O-methylation of A27 in 18S rRNA). Unexpectedly, we found a subset of SNORDs, including SNORD27, in soluble nuclear extract made under native conditions, where fibrillarin was not detected, indicating that a fraction of the SNORD27 RNA likely forms a protein complex different from canonical snoRNAs found in the insoluble nuclear fraction. As part of this previously unidentified complex, SNORD27 regulates the alternative splicing of the transcription factor E2F7 pre-mRNA through direct RNA–RNA interaction without methylating the RNA, likely by competing with U1 small nuclear ribonucleoprotein (snRNP). Furthermore, knockdown of SNORD27 activates previously “silent” exons in several other genes through base complementarity across the entire SNORD27 sequence, not just the antisense boxes. Thus, some SNORDs likely function in both rRNA and pre-mRNA processing, which increases the repertoire of splicing regulators and links both processes

Structure–function analysis of NEET proteins uncovers their role as key regulators of iron and ROS homeostasis in health and disease

AbstractA novel family of 2Fe–2S proteins, the NEET family, was discovered during the last decade in numerous organisms, including archea, bacteria, algae, plant and human; suggesting an evolutionary-conserved function, potentially mediated by their CDGSH Iron–Sulfur Domain. In human, three NEET members encoded by the CISD1–3 genes were identified. The structures of CISD1 (mitoNEET, mNT), CISD2 (NAF-1), and the plant At-NEET uncovered a homodimer with a unique “NEET fold”, as well as two distinct domains: a beta-cap and a 2Fe–2S cluster-binding domain. The 2Fe–2S clusters of NEET proteins were found to be coordinated by a novel 3Cys:1His structure that is relatively labile compared to other 2Fe–2S proteins and is the reason of the NEETs' clusters could be transferred to apo-acceptor protein(s) or mitochondria. Positioned at the protein surface, the NEET's 2Fe–2S's coordinating His is exposed to protonation upon changes in its environment, potentially suggesting a sensing function for this residue. Studies in different model systems demonstrated a role for NAF-1 and mNT in the regulation of cellular iron, calcium and ROS homeostasis, and uncovered a key role for NEET proteins in critical processes, such as cancer cell proliferation and tumor growth, lipid and glucose homeostasis in obesity and diabetes, control of autophagy, longevity in mice, and senescence in plants. Abnormal regulation of NEET proteins was consequently found to result in multiple health conditions, and aberrant splicing of NAF-1 was found to be a causative of the neurological genetic disorder Wolfram Syndrome 2. Here we review the discovery of NEET proteins, their structural, biochemical and biophysical characterization, and their most recent structure–function analyses. We additionally highlight future avenues of research focused on NEET proteins and propose an essential role for NEETs in health and disease. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases

Nutrient-Deprivation Autophagy Factor-1 (NAF-1): Biochemical Properties of a Novel Cellular Target for Anti-Diabetic Drugs

<div><p>Nutrient-deprivation autophagy factor-1 (NAF-1) (synonyms: Cisd2, Eris, Miner1, and Noxp70) is a [2Fe-2S] cluster protein immune-detected both in endoplasmic reticulum (ER) and mitochondrial outer membrane. It was implicated in human pathology (Wolfram Syndrome 2) and in BCL-2 mediated antagonization of Beclin 1-dependent autophagy and depression of ER calcium stores. To gain insights about NAF-1 functions, we investigated the biochemical properties of its 2Fe-2S cluster and sensitivity of those properties to small molecules. The structure of the soluble domain of NAF-1 shows that it forms a homodimer with each protomer containing a [2Fe-2S] cluster bound by 3 Cys and one His. NAF-1 has shown the unusual abilities to transfer its 2Fe-2S cluster to an apo-acceptor protein (followed <i>in vitro</i> by spectrophotometry and by native PAGE electrophoresis) and to transfer iron to intact mitochondria in cell models (monitored by fluorescence imaging with iron fluorescent sensors targeted to mitochondria). Importantly, the drug pioglitazone abrogates NAF-1's ability to transfer the cluster to acceptor proteins and iron to mitochondria. Similar effects were found for the anti-diabetes and longevity-promoting antioxidant resveratrol. These results reveal NAF-1 as a previously unidentified cell target of anti-diabetes thiazolidinedione drugs like pioglitazone and of the natural product resveratrol, both of which interact with the protein and stabilize its labile [2Fe-2S] cluster.</p></div

The 5' untranslated region of the serotonin receptor 2C pre-mRNA generates miRNAs and is expressed in non-neuronal cells

The serotonin receptor 2C (HTR2C) gene encodes a G protein-coupled receptor that is exclusively expressed in neurons. Here, we report that the 5' untranslated region of the receptor pre-mRNA as well as its hosted miRNAs is widely expressed in non-neuronal cell lines. Alternative splicing of HTR2C is regulated by MBII-52. MBII-52 and the neighboring MBII-85 cluster are absent in people with Prader-Willi syndrome, which likely causes the disease. We show that MBII-52 and MBII-85 increase expression of the HTR2C 5' UTR and influence expression of the hosted miRNAs. The data indicate that the transcriptional unit expressing HTR2C is more complex than previously recognized and likely deregulated in Prader-Willi syndrome.This work was supported by NIH RO1 GM083187, P20RR020171 to SS; GM079549 to RS and JS; Binational Science Foundation (BSF), USA-Israel, transformative Grant, #2010508, to SS and RS. EE and AP were supported by the Spanish Ministry of Science with grant BIO2011-23920 and by Sandra Ibarra Foundation for Cancer with grant FSI 2011-03