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Pastore, B.

Publications and source records attributed to Pastore, B..

6 recordsLinked to original sources

Prion Protein Deficiency Results in Synaptic, Neural Network and Behavioral Alterations

The cellular form of the prion protein (PrPC) is known for its involvement in the pathogenesis of prion diseases. Recent research implicates the physiological isoform of PrP in neuronal development, excitability, and synaptic plasticity, as well as in other biological processes. However, its precise function in the development and function of neurons remains poorly understood. Here, we investigated its role during different developmental stages, both in vitro and in vivo, using different PrP knock-out (KO) mouse lines (Prnp-/-). Prion protein KO neurons cultured on microelectrode arrays (MEAs) displayed altered network dynamics compared to wild type cultures, comprising reduced burst frequency, and abnormal spike patterns, indicative of impaired maturation of the synaptic circuitry. These functional alterations were associated with a reduced expression of key presynaptic and postsynaptic proteins, including elements of the SNARE complex and regulators of excitation-inhibition balance. Similar molecular changes were also confirmed in a second Prnp-/- model, suggesting that PrPC is directly involved in these mechanisms regardless of genetic backgrounds. Alterations in neuronal networks were traceable into adulthood: in vivo recordings in adult Prnp-/- mice revealed increased neuronal responses to visual danger stimuli, which correlated with behaviorally increased fear responses to those stimuli. Together, our findings support a critical role for PrPC in the establishment and maintenance of functional neuronal networks, from early developmental stages in vitro to behaviorally mature relevant circuits in vivo, beyond genomic background. These results indicate that PrPC acts as a key regulator of synaptic development and function both in physiological and pathological conditions.

molecular biology↗

Sm-site containing mRNAs can accept Sm-rings and are downregulated in Spinal Muscular Atrophy

Sm-ring assembly is important for the biogenesis, stability, and function of uridine-rich small nuclear RNAs (U snRNAs) involved in pre-mRNA splicing and histone pre-mRNA processing. Sm-ring assembly is cytoplasmic and dependent upon the Sm-site sequence and structural motif, ATP, and Survival motor neuron (SMN) protein complex. While RNAs other than U snRNAs were previously shown to associate with Sm proteins, whether this association follows Sm-ring assembly requirements is unknown. We systematically identified Sm-sites within the human and mouse transcriptomes and assessed whether these sites can accept Sm-rings. In addition to snRNAs, Sm-sites are highly prevalent in the 3 untranslated regions of long messenger RNAs. RNA immunoprecipitation experiments confirm that Sm-site containing mRNAs associate with Sm proteins in the cytoplasm. In modified Sm-ring assembly assays, Sm-site containing RNAs, from either bulk polyadenylated RNAs or those transcribed in vitro, specifically associate with Sm proteins in an Sm-site and ATP-dependent manner. In cell and animal models of Spinal Muscular Atrophy (SMA), mRNAs containing Sm-sites are downregulated, suggesting reduced Sm-ring assembly on these mRNAs may contribute to SMA pathogenesis. Together, this study establishes that Sm-site containing mRNAs can accept Sm-rings and identifies a novel mechanism for Sm proteins in regulation of cytoplasmic mRNAs. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/617433v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@d50f03org.highwire.dtl.DTLVardef@1cca1dforg.highwire.dtl.DTLVardef@1976fcaorg.highwire.dtl.DTLVardef@12c41d9_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

Characterization of ribosome stalling and no-go mRNA decay stimulated by the Fragile X protein, FMRP

Loss of functional fragile X mental retardation protein (FMRP) causes fragile X syndrome (FXS) and is the leading monogenic cause of autism spectrum disorders and intellectual disability. FMRP is most notably a translational repressor and is thought to inhibit translation elongation by stalling ribosomes as FMRP-bound polyribosomes from brain tissue are resistant to puromycin and nuclease treatment. Here, we present data showing that the C-terminal non-canonical RNA-binding domain of FMRP is essential and sufficient to induce puromycin-resistant mRNA*ribosome complexes. Given that stalled ribosomes can stimulate ribosome collisions and no-go mRNA decay (NGD), we tested the ability of FMRP to drive NGD of its target transcripts in neuroblastoma cells. Indeed, FMRP and ribosomal proteins, but not PABPC1, were enriched in isolated nuclease-resistant disomes compared to controls. Using siRNA knockdown and RNA-seq, we identified 16 putative FMRP-mediated NGD substrates, many of which encode proteins involved in neuronal development and function. Increased mRNA stability of the putative substrates was also observed when either FMRP was depleted or NGD was prevented via RNAi. Taken together, these data support that FMRP stalls ribosomes and can stimulate NGD of a select set of transcripts in cells, revealing an unappreciated role of FMRP that would be misregulated in FXS.

biochemistry↗

Pre-piRNA trimming safeguards piRNAs against erroneous targeting by RNA-dependent RNA Polymerase

In animal germ lines, The Piwi/piRNA pathway plays a crucial role in safeguarding genome integrity and promoting fertility. Following transcription from discrete genomic loci, piRNA precursors undergo nucleolytic processing at both 5 and 3 ends. The ribonuclease PARN-1 and its orthologs mediate piRNA 3 trimming in worms, insects and mammals. Yet, the significance of this evolutionarily conserved processing step is not well understood. Employing C. elegans as a model organism, our recent work has demonstrated that 3 trimming protects piRNAs against non-templated nucleotide additions and degradation. In this study, we present an unexpected finding that C. elegans deficient for PARN-1 accumulate a heretofore uncharacterized RNA species termed anti-piRNAs, which are antisense to piRNAs. These anti-piRNAs associate with Piwi proteins and display the propensity for a length of 17-19 nucleotides and 5 guanine and adenine residues. We show that untrimmed pre-piRNAs in parn-1 mutants are modified by the terminal nucleotidyltransferase RDE-3 and erroneously targeted by the RNA-dependent RNA polymerase EGO-1, thereby giving rise to anti-piRNAs. Taken together, our work identifies a previously unknown class of small RNAs upon loss of parn-1 and provides mechanistic insight to activities of RDE-3, EGO-1 and Piwi proteins.

molecular biology↗

Comparative analysis of piRNA sequences, targets and functions in nematodes

Piwi proteins and Piwi-interacting RNAs (piRNAs) are best known for their roles in suppressing transposons and promoting fertility. Yet piRNA biogenesis and its mechanisms of action differ widely between distantly related species. To better understand the evolution of piRNAs, we characterized the piRNA pathway in C. briggsae, a sibling species of the model organism C. elegans. Our analyses define 25,883 piRNA producing-loci in C. briggsae. piRNA sequences in C. briggsae are extremely divergent from their counterparts in C. elegans, yet both species adopt similar genomic organization that drive piRNA expression. By examining production of Piwi-mediated secondary small RNAs, we identified a set of protein-coding genes that are evolutionarily conserved piRNA targets. In contrast to C. elegans, small RNAs targeting ribosomal RNAs or histone transcripts are not hyper-accumulated in C. briggsae Piwi mutants. Instead, we found that transcripts with few introns are prone to small RNA overamplification. Together our work highlights evolutionary conservation and divergence of the nematode piRNA pathway and provides insights into its role in endogenous gene regulation.

genetics↗

Proximity labeling identifies LOTUS domain proteins that promote the formation of perinuclear germ granules in C. elegans

The germline produces gametes that transmit genetic and epigenetic information to the next generation. Maintenance of germ cells and development of gametes require germ granules--well-conserved membraneless and RNA-rich organelles. The composition of germ granules is elusive owing to their dynamic nature and their exclusive expression in the germline. Using C. elegans germ granule, called P granule, as a model system, we employed a proximity-based labeling method in combination with mass spectrometry to comprehensively define its protein components. This set of experiments identified over 200 proteins, many of which contain intrinsically disordered regions. An RNAi-based screen identified factors that are essential for P granule assembly, notably EGGD-1 and EGGD-2, two previously uncharacterized LOTUS-domain proteins. Loss of eggd-1 and eggd-2 results in separation of P granules from nuclear envelope, germline atrophy and reduced fertility. We show that intrinsically disordered regions of EGGD-1 are required to anchor EGGD-1 to the nuclear periphery while its LOTUS domains are required to promote perinuclear localization of P granules. Together, our work expands the repertoire of P granule constituents and provides new insights into the role of LOTUS-domain proteins in germ granule organization.

cell biology↗