| 1. |
- Beneventi, Giulia, et al.
(author)
-
The small Cajal body-specific RNA 15 (SCARNA15) directs p53 and redox homeostasis via selective splicing in cancer cells
- 2021
-
In: NAR Cancer. - : Oxford University Press (OUP). - 2632-8674. ; 3:3
-
Journal article (peer-reviewed)abstract
- Small Cajal body-specific RNAs (scaRNAs) guide post-transcriptional modification of spliceosomal RNA and, while commonly altered in cancer, have poorly defined roles in tumorigenesis. Here, we uncover that SCARNA15 directs alternative splicing (AS) and stress adaptation in cancer cells. Specifically, we find that SCARNA15 guides critical pseudouridylation (Ψ) of U2 spliceosomal RNA to fine-tune AS of distinct transcripts enriched for chromatin and transcriptional regulators in malignant cells. This critically impacts the expression and function of the key tumor suppressors ATRX and p53. Significantly, SCARNA15 loss impairs p53-mediated redox homeostasis and hampers cancer cell sur- vival, motility and anchorage-independent growth. In sum, these findings highlight an unanticipated role for SCARNA15 and Ψ in directing cancer-associated splicing programs.
|
|
| 2. |
- Ciesla, Maciej, et al.
(author)
-
Oncogenic translation directs spliceosome dynamics revealing an integral role for SF3A3 in breast cancer
- 2021
-
In: Molecular Cell. - : Elsevier BV. - 1097-2765. ; 81:7
-
Journal article (peer-reviewed)abstract
- Splicing is a central RNA-based process commonly altered in human cancers; however, how spliceosomal components are co-opted during tumorigenesis remains poorly defined. Here we unravel the core splice factor SF3A3 at the nexus of a translation-based program that rewires splicing during malignant transformation. Upon MYC hyperactivation, SF3A3 levels are modulated translationally through an RNA stem-loop in an eIF3D-dependent manner. This ensures accurate splicing of mRNAs enriched for mitochondrial regulators. Altered SF3A3 translation leads to metabolic reprogramming and stem-like properties that fuel MYC tumorigenic potential in vivo. Our analysis reveals that SF3A3 protein levels predict molecular and phenotypic features of aggressive human breast cancers. These findings unveil a post-transcriptional interplay between splicing and translation that governs critical facets of MYC-driven oncogenesis.
|
|
| 3. |
- Georgakopoulos-Soares, Ilias, et al.
(author)
-
Alternative splicing modulation by G-quadruplexes
- 2022
-
In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13:1
-
Journal article (peer-reviewed)abstract
- Alternative splicing is central to metazoan gene regulation, but the regulatory mechanisms are incompletely understood. Here, we show that G-quadruplex (G4) motifs are enriched ~3-fold near splice junctions. The importance of G4s in RNA is emphasised by a higher enrichment for the non-template strand. RNA-seq data from mouse and human neurons reveals an enrichment of G4s at exons that were skipped following depolarisation induced by potassium chloride. We validate the formation of stable RNA G4s for three candidate splice sites by circular dichroism spectroscopy, UV-melting and fluorescence measurements. Moreover, we find that sQTLs are enriched at G4s, and a minigene experiment provides further support for their role in promoting exon inclusion. Analysis of >1,800 high-throughput experiments reveals multiple RNA binding proteins associated with G4s. Finally, exploration of G4 motifs across eleven species shows strong enrichment at splice sites in mammals and birds, suggesting an evolutionary conserved splice regulatory mechanism.
|
|
| 4. |
- Guzzi, Nicola, et al.
(author)
-
Pseudouridine-modified tRNA fragments repress aberrant protein synthesis and predict leukaemic progression in myelodysplastic syndrome
- 2022
-
In: Nature Cell Biology. - : Springer Science and Business Media LLC. - 1465-7392 .- 1476-4679. ; 24:3, s. 299-306
-
Journal article (peer-reviewed)abstract
- Transfer RNA-derived fragments (tRFs) are emerging small noncoding RNAs that, although commonly altered in cancer, have poorly defined roles in tumorigenesis1. Here we show that pseudouridylation (Ψ) of a stem cell-enriched tRF subtype2, mini tRFs containing a 5′ terminal oligoguanine (mTOG), selectively inhibits aberrant protein synthesis programmes, thereby promoting engraftment and differentiation of haematopoietic stem and progenitor cells (HSPCs) in patients with myelodysplastic syndrome (MDS). Building on evidence that mTOG-Ψ targets polyadenylate-binding protein cytoplasmic 1 (PABPC1), we employed isotope exchange proteomics to reveal critical interactions between mTOG and functional RNA-recognition motif (RRM) domains of PABPC1. Mechanistically, this hinders the recruitment of translational co-activator PABPC1-interacting protein 1 (PAIP1)3 and strongly represses the translation of transcripts sharing pyrimidine-enriched sequences (PES) at the 5′ untranslated region (UTR), including 5′ terminal oligopyrimidine tracts (TOP) that encode protein machinery components and are frequently altered in cancer4. Significantly, mTOG dysregulation leads to aberrantly increased translation of 5′ PES messenger RNA (mRNA) in malignant MDS-HSPCs and is clinically associated with leukaemic transformation and reduced patient survival. These findings define a critical role for tRFs and Ψ in difficult-to-treat subsets of MDS characterized by high risk of progression to acute myeloid leukaemia (AML).
|
|
| 5. |
- Guzzi, Nicola, et al.
(author)
-
Pseudouridylation of tRNA-Derived Fragments Steers Translational Control in Stem Cells
- 2018
-
In: Cell. - : Elsevier BV. - 0092-8674 .- 1097-4172. ; 173:5, s. 26-1216
-
Journal article (peer-reviewed)abstract
- Pseudouridylation (Ψ) is the most abundant and widespread type of RNA epigenetic modification in living organisms; however, the biological role of Ψ remains poorly understood. Here, we show that a Ψ-driven posttranscriptional program steers translation control to impact stem cell commitment during early embryogenesis. Mechanistically, the Ψ “writer” PUS7 modifies and activates a novel network of tRNA-derived small fragments (tRFs) targeting the translation initiation complex. PUS7 inactivation in embryonic stem cells impairs tRF-mediated translation regulation, leading to increased protein biosynthesis and defective germ layer specification. Remarkably, dysregulation of this posttranscriptional regulatory circuitry impairs hematopoietic stem cell commitment and is common to aggressive subtypes of human myelodysplastic syndromes. Our findings unveil a critical function of Ψ in directing translation control in stem cells with important implications for development and disease. Translational control in stem cells is orchestrated by pseudouridylation of specific tRNA-derived fragments, impacting stem cell commitment during key developmental processes.
|
|
| 6. |
- Johansson, Therese B., et al.
(author)
-
Sub-Nanomolar Detection of Oligonucleotides Using Molecular Beacons Immobilized on Lightguiding Nanowires
- 2024
-
In: Nanomaterials. - 2079-4991. ; 14:5
-
Journal article (peer-reviewed)abstract
- The detection of oligonucleotides is a central step in many biomedical investigations. The most commonly used methods for detecting oligonucleotides often require concentration and amplification before detection. Therefore, developing detection methods with a direct read-out would be beneficial. Although commonly used for the detection of amplified oligonucleotides, fluorescent molecular beacons have been proposed for such direct detection. However, the reported limits of detection using molecular beacons are relatively high, ranging from 100 nM to a few µM, primarily limited by the beacon fluorescence background. In this study, we enhanced the relative signal contrast between hybridized and non-hybridized states of the beacons by immobilizing them on lightguiding nanowires. Upon hybridization to a complementary oligonucleotide, the fluorescence from the surface-bound beacon becomes coupled in the lightguiding nanowire core and is re-emitted at the nanowire tip in a narrower cone of light compared with the standard 4π emission. Prior knowledge of the nanowire positions allows for the continuous monitoring of fluorescence signals from each nanowire, which effectively facilitates the discrimination of signals arising from hybridization events against background signals. This resulted in improved signal-to-background and signal-to-noise ratios, which allowed for the direct detection of oligonucleotides at a concentration as low as 0.1 nM.
|
|
| 7. |
- Pereira, Luis Alberto, et al.
(author)
-
Long 3’UTR of Nurr1 mRNAs is targeted by miRNAs in mesencephalic dopamine neurons
- 2017
-
In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 12:11
-
Journal article (peer-reviewed)abstract
- The development of mesencephalic dopamine neurons and their survival later in life requires the continuous presence of the transcription factor Nurr1 (NR4A2). Nurr1 belongs to the nuclear receptors superfamily. However, it is an orphan member that does not require a ligand to regulate the transcription of its target genes. Therefore, controlling the expression of Nurr1 is an important manner to control its function. Several reports have shown that microRNAs (miRNAs) regulate Nurr1 expression. However, Nurr1 has several splicing variants, posing the question what variants are subjected to miRNA regulation. In this work, we identified a long 3’UTR variant of rat Nurr1 mRNA. We used bioinformatics analysis to identify miRNAs with the potential to regulate Nurr1 expression. Reporter assays performed with the luciferase gene fused to the short (658 bp) or long (1,339 bp) 3’UTR of rat Nurr1 mRNAs, showed that miR-93, miR-204 and miR-302d selectively regulate the mRNA with the longest 3’UTR. We found that the longest variant of Nurr1 mRNA expresses in the rat mesencephalon as assessed by PCR. The transfection of rat mesencephalic neurons with mixed miR-93, miR-204 and miR-302d resulted in a significant reduction of Nurr1 protein levels. In conclusion, Nurr1 mRNA variant with the longest 3’UTR undergoes a specific regulation by miRNAs. It is discussed the importance of fine-tuning Nurr1 protein levels in mesencephalic dopamine neurons.
|
|
| 8. |
- Volpati, Diogo, et al.
(author)
-
Monitoring the Intracellular Fate of Molecular Beacons : The Challenge of False Positive Signals
-
In: Advanced NanoBiomed Research. - 2699-9307.
-
Journal article (peer-reviewed)abstract
- Molecular beacons (MBs) have been used on surfaces for detecting oligonucleotides. Attempts to use them intracellularly for monitoring mRNA content have been made, however, without any clear conclusion regarding the reliability of the method, mainly due to false positive signals. To reach an understanding of the intracellular fate of MBs, a critical question remains: how long after MB delivery and where in the cell does a false positive signal appear? To answer that question, the MB delivery method should allow for a time-stamped synchronized delivery of MBs to multiple cells, resulting in MBs being distributed in the cytosol immediately after delivery. Herein, nanostraws are used to inject MBs targeting insulin (Ins1) mRNA directly in the cytosol of clonal beta-cells, and the evolution of the MB fluorescence in time and space is monitored. The results show an MB translocation to the nucleus, where MBs are degraded or where they open nonspecifically, before the fluorophore alone is expelled back from the nucleus to the cytosol. The signal translocation to the nucleus and back to the cytosol is faster when scrambled MBs are used. The results shed light on the intracellular fate of MBs and highlight the short time scales before false positive signals become predominant.
|
|
