Alessandro Fatica

Long non-coding RNAs: new players in cell differentiation and development

Genomes of multicellular organisms are characterized by the pervasive expression of different types of non-coding RNAs (ncRNAs). Long ncRNAs (lncRNAs) belong to a novel heterogeneous class of ncRNAs that includes thousands of different species. lncRNAs have crucial roles in gene expression control during both developmental and differentiation processes, and the number of lncRNA species increases in genomes of developmentally complex organisms, which highlights the importance of RNA-based levels of control in the evolution of multicellular organisms. In this Review, we describe the function of lncRNAs in developmental processes, such as in dosage compensation, genomic imprinting, cell differentiation and organogenesis, with a particular emphasis on mammalian development.

A Minicircuitry Comprised of MicroRNA-223 and Transcription Factors NFI-A and C/EBPα Regulates Human Granulopoiesis

MicroRNAs play important roles in cell differentiation by acting as translational inhibitors of specific target genes. Here we show that human granulocytic differentiation is controlled by a regulatory circuitry involving miR-223 and two transcriptional factors, NFI-A and C/EBPalpha. The two factors compete for binding to the miR-223 promoter: NFI-A maintains miR-223 at low levels, whereas its replacement by C/EBPalpha, following retinoic acid (RA)-induced differentiation, upregulates miR-223 expression. The competition by C/EBPalpha and the granulocytic differentiation are favored by a negative-feedback loop in which miR-223 represses NFI-A translation. In line with this, both RNAi against NFI-A and ectopic expression of miR-223 in acute promyelocytic leukemia (APL) cells enhance differentiation, whereas miR-223 knockdown inhibits the differentiation response to RA. Altogether, our data indicate that miR-223 plays a crucial role during granulopoiesis and point to the NFI-A repression as an important molecular pathway mediating gene reprogramming in this cell lineage.

The interplay between the master transcription factor PU.1 and miR-424 regulates human monocyte/macrophage differentiation

We describe a pathway by which the master transcription factor PU.1 regulates human monocyte/macrophage differentiation. This includes miR-424 and the transcriptional factor NFI-A. We show that PU.1 and these two components are interlinked in a finely tuned temporal and regulatory circuitry: PU.1 activates the transcription of miR-424, and this up-regulation is involved in stimulating monocyte differentiation through miR-424-dependent translational repression of NFI-A. In turn, the decrease in NFI-A levels is important for the activation of differentiation-specific genes such as M-CSFr. In line with these data, both RNAi against NFI-A and ectopic expression of miR-424 in precursor cells enhance monocytic differentiation, whereas the ectopic expression of NFI-A has an opposite effect. The interplay among these three components was demonstrated in myeloid cell lines as well as in human CD34+ differentiation. These data point to the important role of miR-424 and NFI-A in controlling the monocyte/macrophage differentiation program.

Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis

Summary Circular RNAs (circRNAs) constitute a family of transcripts with unique structures and still largely unknown functions. Their biogenesis, which proceeds via a back-splicing reaction, is fairly well characterized, whereas their role in the modulation of physiologically relevant processes is still unclear. Here we performed expression profiling of circRNAs during in vitro differentiation of murine and human myoblasts, and we identified conserved species regulated in myogenesis and altered in Duchenne muscular dystrophy. A high-content functional genomic screen allowed the study of their functional role in muscle differentiation. One of them, circ-ZNF609, resulted in specifically controlling myoblast proliferation. Circ-ZNF609 contains an open reading frame spanning from the start codon, in common with the linear transcript, and terminating at an in-frame STOP codon, created upon circularization. Circ-ZNF609 is associated with heavy polysomes, and it is translated into a protein in a splicing-dependent and cap-independent manner, providing an example of a protein-coding circRNA in eukaryotes.

Processing of the intron-encoded U16 and U18 snoRNAs: The conserved C and D boxes control both the processing reaction and the stability of the mature snoRNA

A novel class of small nucleolar RNAs (snoRNAs), encoded in introns of protein coding genes and originating from processing of their precursor molecules, has recently been described. The L1 ribosomal protein (r‐protein) gene of Xenopus laevis and its human homologue contain two snoRNAs, U16 and U18. It has been shown that these snoRNAs are excised from their intron precursors by endonucleolytic cleavage and that their processing is alternative to splicing. Two sequences, internal to the snoRNA coding region, have been identified as indispensable for processing the conserved boxes C and D. Competition experiments have shown that these sequences interact with diffusible factors which can bind both the pre‐mRNA and the mature U16 snoRNA. Fibrillarin, which is known to associate with complexes formed on C and D boxes of other snoRNAs, is found in association with mature U16 RNA, as well as with its precursor molecules. This fact suggests that the complex formed on the pre‐mRNA remains bound to U16 throughout all the processing steps. We also show that the complex formed on the C and D boxes is necessary to stabilize mature snoRNA.

A Feedforward Regulatory Loop between HuR and the Long Noncoding RNA linc-MD1 Controls Early Phases of Myogenesis

Summary The muscle-specific long noncoding RNA linc-MD1 was shown to be expressed during early phases of muscle differentiation and to trigger the switch to later stages by acting as a sponge for miR-133 and miR-135. Notably, linc-MD1 is also the host transcript of miR-133b, and their biogenesis is mutually exclusive. Here, we describe that this alternative synthesis is controlled by the HuR protein, which favors linc-MD1 accumulation through its ability to bind linc-MD1 and repress Drosha cleavage. We show that HuR is under the repressive control of miR-133 and that the sponging activity of linc-MD1 consolidates HuR expression in a feedforward positive loop. Finally, we show that HuR also acts in the cytoplasm, reinforcing linc-MD1 sponge activity by cooperating for miRNA recruitment. An increase in miR-133 synthesis, mainly from the two unrelated miR-133a coding genomic loci, is likely to trigger the exit from this circuitry and progression to later differentiation stages.

Heterochromatin Protein 1 (HP1a) Positively Regulates Euchromatic Gene Expression through RNA Transcript Association and Interaction with hnRNPs in Drosophila

Heterochromatin Protein 1 (HP1a) is a well-known conserved protein involved in heterochromatin formation and gene silencing in different species including humans. A general model has been proposed for heterochromatin formation and epigenetic gene silencing in different species that implies an essential role for HP1a. According to the model, histone methyltransferase enzymes (HMTases) methylate the histone H3 at lysine 9 (H3K9me), creating selective binding sites for itself and the chromodomain of HP1a. This complex is thought to form a higher order chromatin state that represses gene activity. It has also been found that HP1a plays a role in telomere capping. Surprisingly, recent studies have shown that HP1a is present at many euchromatic sites along polytene chromosomes of Drosophila melanogaster, including the developmental and heat-shock-induced puffs, and that this protein can be removed from these sites by in vivo RNase treatment, thus suggesting an association of HP1a with the transcripts of many active genes. To test this suggestion, we performed an extensive screening by RIP-chip assay (RNA–immunoprecipitation on microarrays), and we found that HP1a is associated with transcripts of more than one hundred euchromatic genes. An expression analysis in HP1a mutants shows that HP1a is required for positive regulation of these genes. Cytogenetic and molecular assays show that HP1a also interacts with the well known proteins DDP1, HRB87F, and PEP, which belong to different classes of heterogeneous nuclear ribonucleoproteins (hnRNPs) involved in RNA processing. Surprisingly, we found that all these hnRNP proteins also bind heterochromatin and are dominant suppressors of position effect variegation. Together, our data show novel and unexpected functions for HP1a and hnRNPs proteins. All these proteins are in fact involved both in RNA transcript processing and in heterochromatin formation. This suggests that, in general, similar epigenetic mechanisms have a significant role on both RNA and heterochromatin metabolisms.

Purified Box C/D snoRNPs Are Able To Reproduce Site-Specific 2′-O-Methylation of Target RNA In Vitro

ABSTRACT Small nucleolar RNAs (snoRNAs) are associated in ribonucleoprotein particles localized to the nucleolus (snoRNPs). Most of the members of the box C/D family function in directing site-specific 2′-O-methylation of substrate RNAs. Although the selection of the target nucleotide requires the antisense element and the conserved box D or D′ of the snoRNA, the methyltransferase activity is supposed to reside in one of the protein components. Through protein tagging of a snoRNP-specific factor, we purified to homogeneity box C/D snoRNPs from the yeast Saccharomyces cerevisiae. Mass spectrometric analysis demonstrated the presence of Nop1p, Nop58p, Nop56p, and Snu13p as integral components of the particle. We show that purified snoRNPs are able to reproduce the site-specific methylation pattern on target RNA and that the predicted S-adenosyl-l-methionine-binding region of Nop1p is responsible for the catalytic activity.

Microarray detection of novel nuclear RNA substrates for the exosome.

Microarray analyses were performed on yeast strains mutant for the nuclear‐specific exosome components Rrp6p and Rrp47p/Lrp1p or the core component Rrp41p/Ski6p, at permissive temperature and following transfer to 37 °C. 339 mRNAs showed clearly altered expression levels, with an unexpectedly high degree of heterogeneity in the different exosome mutants. In contrast, no clear alterations were seen in strains lacking the cytoplasmic exosome component Ski7p. 27 mRNAs that were overexpressed in each strain defective in the nuclear exosome are good candidates for regulation by nuclear turnover. These included the mRNA for the autoregulated RNA‐binding protein Nrd1p. Northern and primer extension analyses confirmed the elevated NRD1 mRNA levels in exosome mutants, and revealed the accumulation of truncated 5′ fragments of the mRNA. These contain a predicted Nrd1p‐binding site, potentially sequestering the protein and disrupting its autoregulation. Several genes located immediately downstream of independently transcribed snoRNA genes were overexpressed in exosome mutants, presumably due to stabilization of the products of transcription termination read‐through. Further analyses indicated that many snoRNA and snRNA genes are inefficiently terminated, but read‐through transcripts into downstream ORFs are normally rapidly degraded by the exosome. Copyright

The Cotranscriptional Assembly of snoRNPs Controls the Biosynthesis of H/ACA snoRNAs in Saccharomyces cerevisiae

ABSTRACT The carboxy-terminal domain (CTD) of RNA polymerase II large subunit acts as a platform to assemble the RNA processing machinery in a controlled way throughout the transcription cycle. In yeast, recent findings revealed a physical connection between phospho-CTD, generated by the Ctk1p kinase, and protein factors having a function in small nucleolar RNA (snoRNA) biogenesis. The snoRNAs represent a large family of polymerase II noncoding transcripts that are associated with highly conserved polypeptides to form stable ribonucleoprotein particles (snoRNPs). In this work, we have studied the biogenesis of the snoRNPs belonging to the box H/ACA class. We report that the assembly factor Naf1p and the core components Cbf5p and Nhp2p are recruited on H/ACA snoRNA genes very early during transcription. We also show that the cotranscriptional recruitment of Naf1p and Cbf5p is Ctk1p dependent and that Ctk1p and Cbf5p are required for preventing the readthrough into the snoRNA downstream genes. All these data suggest that proper cotranscriptional snoRNP assembly controls 3′-end formation of snoRNAs and, consequently, the release of a functional particle.