A novel partial de novo duplication of JARID2 gene causing a neurodevelopmental phenotype

Publisher Copyright: © 2022 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.Background: Deletions covering the entire or partial JARID2 gene as well as pathogenic single nucleotide variants leading to haploinsufficiency of JARID2 have recently been shown to cause a clinically distinct neurodevelopmental syndrome. Here, we present a previously undescribed partial de novo duplication of the JARID2 gene in a patient displaying features similar to those of patients with JARID2 loss-of-function variants. Case report: The index patient presents with abnormalities in gross motor skills and speech development as well as neuropsychiatric disorders. The patient has markedly dark infraorbital circles and slightly prominent supraorbital ridges.Whole-genome sequencing and array comparative genomic hybridization revealed a novel disease-causing variant type, a partial tandem duplication of JARID2, covering the exons 1–7. Furthermore, RNA sequencing validated the increased expression of these exons. Expression alterations were also detected in target genes of the PRC2 complex, in which JARID2 acts as an essential member. Conclusion: Our data add to the variety of different pathogenic variants associated with JARID2 neurodevelopmental syndrome.Peer reviewe

Virus-Encapsulated DNA Origami Nanostructures for Cellular Delivery

Peer reviewe

Global analysis of the nuclear processing of transcripts with unspliced U12-type introns by the exosome

Peer reviewe

Asymmetric gait results in symmetric muscular demands in affected versus unaffected side ankle and knee extensors in hemiplegic CP children

Children with hemiplegic cerebral palsy (CP) exhibit asymmetric gait due to compromised neuromuscular function of the affected lower limb [1]. According to a longstanding theory, natural gait patterns coincide with the minimal metabolic cost [2]. We hypothesized that biomechanical constraints may also play a critical role in determining the way humans move; for example, leading to compensation strategies that help to accommodate compromised paretic limb force production. To test this, we examined gait pattern asymmetries and relative muscle efforts of the ankle and knee extensors between affected versus unaffected lower limbs in hemiplegic CP children.nonPeerReviewe

Evolutionarily conserved exon definition interactions with U11 snRNP mediate alternative splicing regulation on U11–48K and U11/U12–65K genes

<div><p>Many splicing regulators bind to their own pre-mRNAs to induce alternative splicing that leads to formation of unstable mRNA isoforms. This provides an autoregulatory feedback mechanism that regulates the cellular homeostasis of these factors. We have described such an autoregulatory mechanism for two core protein components, U11–48K and U11/U12–65K, of the U12-dependent spliceosome. This regulatory system uses an atypical splicing enhancer element termed USSE (U11 snRNP-binding splicing enhancer), which contains two U12-type consensus 5′ splice sites (5′ss). Evolutionary analysis of the USSE element from a large number of animal and plant species indicate that USSE sequence must be located 25–50 nt downstream from the target 3′ splice site (3′ss). Together with functional evidence showing a loss of USSE activity when this distance is reduced and a requirement for RS-domain of U11–35K protein for 3′ss activation, our data suggests that U11 snRNP bound to USSE uses exon definition interactions for regulating alternative splicing. However, unlike standard exon definition where the 5′ss bound by U1 or U11 will be subsequently activated for splicing, the USSE element functions similarly as an exonic splicing enhancer and is involved only in upstream splice site activation but does not function as a splicing donor. Additionally, our evolutionary and functional data suggests that the function of the 5′ss duplication within the USSE elements is to allow binding of two U11/U12 di-snRNPs that stabilize each others' binding through putative mutual interactions.</p></div

<i>U11/U12-65K</i> transcripts with a long 3′UTR are associated with transcriptional read-through.

<p><b>(A)</b> Following transfection with mock or block morpholinos in HEK293 cells, RT-PCR was performed to detect both <i>65K</i> isoforms simultaneously, using a reverse primer binding at the canonical terminal exon (upper panel), or located in the IE (lower panel). Amplicons were separated on a 1% agarose gel. Endoporter transfection reagent was used for the (-) control sample. The marker (M) is Generuler (Thermo Scientific: #SM0331). <b>(B)</b> qRT-PCR analysis of IE expression level for morpholino transfected samples normalized against total <i>65K</i> expression. Error bars represent the standard deviation of 3 biological replicates and the asterisk indicates p-value < 0.05 in a two-tailed Student′s t-test in (B), (D), (F) and (G). <b>(C)</b> Upper panel. Schematics of the reporter plasmid construction. Shaded boxes depict <i>65K</i> sequences cloned into pGL4.13. Lower panel indicate mutations of the putative poly(A) sites (in bold and underlined) of the <i>65K</i> gene to generate the pAdel reporter construct. <b>(D)</b> CHO cells were transfected with wt and pAdel reporter constructs and construct <i>65K</i> long-3′UTR and short-3′UTR splicing isoforms were quantified by qPCR. Fold change is relative to the wt construct <i>65K</i> short-3′UTR splicing isoform. <b>(E)</b> Multiplex (upper panel) and normal RT-PCR of the samples analyzed in (D) were performed and amplicons separated on 1.3% agarose gel. Primers (shown as arrows) were designed to quantify construct <i>65K</i> 3′UTR splicing isoform ratio (upper panel) and read-through and IE splicing of construct <i>65K</i> short- and long-3′UTR splicing isoform transcripts. Dashed lines indicate an exon-spanning primer and an asterisk a construct-specific isoform (splicing event could not be found from endogenous <i>65K</i> gene) skipping the short-3′UTR specific exon and directly splicing towards to IE. <b>(F)</b> qPCR quantification of construct <i>65K</i> 3′UTR splicing isoform transcripts that undergo IE splicing from samples analyzed in (E). <b>(G)</b> RT-PCR analysis of expression of short- and long-3′UTR splicing isoform transcripts that read-through past the poly(A) site but do not splice to the IE (RT transcripts) from samples analyzed in (E). Fold change is relative to the wt RT transcript expression and samples were normalized against total construct <i>65K</i> short- or long-3′UTR splicing isoform levels.</p

Model for <i>65K</i> regulation: Alternative exon definition interactions in the <i>65K</i> 3′UTR determine the choice between nuclear export and nuclear retention.

<p>Alternative splicing in the <i>65K</i> gene generates either a short-3′UTR splicing isoform (productive) or a long-3′UTR splicing isoform (non-productive). For the long-3′UTR isoform, aberrant 3′-end processing together with bound U11 snRNP (or U11/U12 di-snRNP) as a retention factor provide a dual mechanism for nuclear retention. For the short-3′UTR isoform, optimal terminal exon definition interactions overcome U1-mediated inhibition of CP to ensure efficient nuclear export.</p

Nuclear retention of <i>65K</i> long-3′UTR splicing isoform requires USSE-directed splicing in the 3′UTR.

<p><b>(A)</b> HeLa cells were transfected with reporter constructs depicted in the top panels. Following cellular fractionation, the splicing pattern in the 3′UTR was analyzed by multiplex RT-PCR for the constructs and the endogenous <i>65K</i>. Arrows indicate primer location. T is total, C is cytoplasmic and N is nuclear fraction. Error bars represent standard deviation for 3 biological replicates and the asterisk indicates p-value < 0.05 in two-tailed Student′s t-test. in (A) and (C). <b>(B)</b> Schematics of the reporters used in (C) and (D): 4 BoxB hairpin (HP), wild-type (WT), and the CT+67/GA construct. For the CT+6/7GA construct, mutations (in bold) were made in both U12 5′ss motifs of the USSE. <b>(C)</b> HeLa cells were transfected with depicted reporters with, or without co-transfection of U11 snRNA construct carrying compensatory mutations. After cellular fractionation, distribution of construct-derived long and short-3′UTR splicing isoforms was assayed through RT-PCR. Endogenous <i>65K</i> isoform cellular distribution served as fractionation quality control. T is total, C is cytoplasmic and N is nuclear fraction. <b>(D)</b> HeLa cells were transfected with depicted reporters with, or without co-transfection of λN peptide-fused U1-70K expression construct. After cellular fractionation, distribution of construct-derived long and short-3′UTR splicing isoform was assayed through RT-PCR. Endogenous <i>65K</i> isoform cellular distribution served as fractionation quality control. T is total, C is cytoplasmic and N is nuclear fraction.</p

Dynamic regulation of <i>65K</i> alternative splicing in developing neuronal cells leads to increase in long-3′UTR isoform expression and transcriptional read-through.

<p><b>(A)</b> Model of alternative splicing in the <i>65K</i> gene. Binding of U11/U12 di-snRNP to USSE activates alternative splicing to form an isoform with a long 3′UTR. Exons are in blue and the 3′UTR in yellow. <b>(B)</b> Sashimi plots of RNAseq data in the 3′ end of the <i>Rnpc3</i> locus (<i>U11/U12-65K</i>) gene from mouse stem cells (ESC: embryonic stem cells, NSC: neural stem cells), radial glia-type (RG) and glutamatergic neurons (GN) of increasing maturity (DIV1-28). Junction reads are plotted as arcs with the number indicating number of exon-spanning reads. <b>(C)</b> Sashimi plot of RNAseq reads in region downstream of the <i>Rnpc3</i> (<i>U11/U12-65K</i>) gene and in the <i>Amy1</i> gene. <b>(D)</b> RT-PCR analysis of <i>Rnpc3</i> (<i>65K</i>) isoform ratio and <i>Rnpc3-Amy1</i> conjoined transcripts (upper section) and Western blot analysis (lower section) in mouse cortical neural stem cells and cortical neurons cells. The identities of the amplicons are indicated on left with arrows showing the location of primers. Immunoprecipitation followed by Western blots were performed using a U11/U12 65K antibody (Proteintech, 25820-1-AP) and shown in the panel labeled as "IP". For normalization, p44/42 MAPK (Erk1/2) (Cell Signaling, #4695) were probed in the panel labeled "Load" that represents the sample prior IP. Asterisk indicates the IgG heavy chain. <b>(E)</b> RT-PCR quantification of the indicated isoforms from samples shown in (D). Error bars represent the standard deviation of 3 biological replicates and the asterisk indicates p-value < 0.05 in two-tailed Student′s t-test in (E), (F) and (G). <b>(F)</b> Fold-change quantification by RT-PCR of conjoined <i>Rnpc3-Amy1</i> transcripts from samples shown in (D). Samples were normalized with total <i>65K</i> (<i>Rnpc3)</i> expression. <b>(G)</b> Quantification of the relative U11/U12-65K protein levels from Western blot samples shown in (D). Samples were normalized with p44 MAPK (Erk1) expression. <b>(H)</b> Sashimi plot of ENCODE RNAseq reads in the <i>RNPC3</i> (<i>U11/U12-65K</i>) gene and <i>AMY2B</i> gene from human cerebellum and HeLa S3 cells. Junction reads are plotted as arcs with the number indicating number of reads. Arrows indicate position of USSE, 5’ss, pA (poly(A) site) and IE (intergenic exon).</p