Replication origins drive genetic and phenotypic variation in humans

DNA replication starts with the activation of the replicative helicases, polymerases and associated factors at thousands of origins per S-phase. Due to local torsional constraints generated during licensing and the switch between polymerases of distinct fidelity and proofreading ability following firing, origin activation has the potential to induce DNA damage and mutagenesis. However, whether sites of replication initiation exhibit a specific mutational footprint has not yet been established. Here we demonstrate that mutagenesis is increased at early and highly efficient origins. The elevated mutation rate observed at these sites is caused by two distinct mutational processes consistent with formation of DNA breaks at the origin itself and local error-prone DNA synthesis in the immediate vicinity of the origin. We demonstrate that these replication-dependent mutational processes create the skew in base composition observed at human replication origins. Further, we show that mutagenesis associated with replication initiation exerts an influence on phenotypic diversity in human populations disproportionate to the origins genomic footprint: by increasing mutational loads at gene promoters and splice junctions the presence of an origin influences both gene expression and mRNA isoform usage. These findings have important implications for our understanding of the mutational processes that sculpt the human genome.

Identification of an mRNA isoform switch for HNRNPA1 in breast cancers

Roles of HNRNPA1 are beginning to emerge in cancers; however, mechanisms causing deregulation of HNRNPA1 function remain elusive. Here, we describe an isoform switch between the 3′-UTR isoforms of HNRNPA1 in breast cancers. We show that the dominantly expressed isoform in mammary tissue has a short half-life. In breast cancers, this isoform is downregulated in favor of a stable isoform. The stable isoform is expressed more in breast cancers, and more HNRNPA1 protein is synthesized from this isoform. High HNRNPA1 protein levels correlate with poor survival in patients. In support of this, silencing of HNRNPA1 causes a reversal in neoplastic phenotypes, including proliferation, clonogenic potential, migration, and invasion. In addition, silencing of HNRNPA1 results in the downregulation of microRNAs that map to intragenic regions. Among these miRNAs, miR-21 is known for its transcriptional upregulation in breast and numerous other cancers. Altogether, the cancer-specific isoform switch we describe here for HNRNPA1 emphasizes the need to study gene expression at the isoform level in cancers to identify novel cases of oncogene activation.

Tissue-specific expression of p73 and p63 isoforms in human tissues

Abstractp73 and p63 are members of the p53 family that exhibit overlapping and distinct functions in development and homeostasis. The evaluation of p73 and p63 isoform expression across human tissue can provide greater insight to the functional interactions between family members. We determined the mRNA isoform expression patterns of TP73 and TP63 across a panel of 36 human tissues and protein expression within the highest-expressing tissues. TP73 and TP63 expression significantly correlated across tissues. In tissues with concurrent mRNA expression, nuclear co-expression of both proteins was observed in a majority of cells. Using GTEx data, we quantified p73 and p63 isoform expression in human tissue and identified that the α-isoforms of TP73 and TP63 were the predominant isoform expressed in nearly all tissues. Further, we identified a previously unreported p73 mRNA product encoded by exons 4 to 14. In sum, these data provide the most comprehensive tissue-specific atlas of p73 and p63 protein and mRNA expression patterns in human and murine samples, indicating coordinate expression of these transcription factors in the majority of tissues in which they are expressed.

First person – Matthew Zdradzinski

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Matthew Zdradzinski is co-first author on ‘Selective axonal translation of the mRNA isoform encoding prenylated Cdc42 supports axon growth’, published in JCS. Matthew is a PhD Student in the lab of Jeffery Twiss at the Department of Biological Sciences, University of South Carolina, Columbia, SC, where he is interested in neurobiology, focused around mRNA localization and its effects on axon growth, development and regeneration.

Selective axonal translation of the mRNA isoform encoding prenylated Cdc42 supports axon growth

ABSTRACT The small Rho-family GTPase Cdc42 has long been known to have a role in cell motility and axon growth. The eukaryotic Ccd42 gene is alternatively spliced to generate mRNAs with two different 3′ untranslated regions (UTRs) that encode proteins with distinct C-termini. The C-termini of these Cdc42 proteins include CaaX and CCaX motifs for post-translational prenylation and palmitoylation, respectively. Palmitoyl-Cdc42 protein was previously shown to contribute to dendrite maturation, while the prenyl-Cdc42 protein contributes to axon specification and its mRNA was detected in neurites. Here, we show that the mRNA encoding prenyl-Cdc42 isoform preferentially localizes into PNS axons and this localization selectively increases in vivo during peripheral nervous system (PNS) axon regeneration. Functional studies indicate that prenyl-Cdc42 increases axon length in a manner that requires axonal targeting of its mRNA, which, in turn, needs an intact C-terminal CaaX motif that can drive prenylation of the encoded protein. In contrast, palmitoyl-Cdc42 has no effect on axon growth but selectively increases dendrite length. Together, these data show that alternative splicing of the Cdc42 gene product generates an axon growth promoting, locally synthesized prenyl-Cdc42 protein. This article has an associated First Person interview with one of the co-first authors of the paper.

A conserved role for the ALS-linked splicing factor SFPQ in repression of pathogenic cryptic last exons

The RNA-binding protein SFPQ plays an important role in neuronal development and has been associated with several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer’s disease. Here, we report that loss of sfpq leads to premature termination of multiple transcripts due to widespread activation of previously unannotated cryptic last exons (CLEs). These SFPQ-inhibited CLEs appear preferentially in long introns of genes with neuronal functions and can dampen gene expression outputs and/or give rise to short peptides interfering with the normal gene functions. We show that one such peptide encoded by the CLE-containing epha4b mRNA isoform is responsible for neurodevelopmental defects in the sfpq mutant. The uncovered CLE-repressive activity of SFPQ is conserved in mouse and human, and SFPQ-inhibited CLEs are found expressed across ALS iPSC-derived neurons. These results greatly expand our understanding of SFPQ function and uncover a gene regulation mechanism with wide relevance to human neuropathologies.

Same same but different : a comprehensive functional analysis of SRSF3 and SRSF7 in the regulation of alternative polyadenylation within the 3’ untranslated region

Das klassische zentrale Dogma der Biologie beschreibt die Synthese funktionaler Proteine basierend auf den Informationen, die in der DNA kodiert sind. In einem notwendigen Zwischenschritt wird zunächst die entsprechende DNA-Sequenz in ein messenger-RNA (mRNA) Molekül abgeschrieben (transkribiert), bevor diese RNA-Sequenz durch Ribosomen in das finale Protein übersetzt (translatiert) werden kann. In Eukaryoten sind die Transkription und Translation durch eine Kompartimentierung der Zelle in den Zellkern und das Zytosol örtlich und zeitlich voneinander getrennt. Diese Trennung ermöglicht eine eingehende Qualitätskontrolle der gereiften mRNA im Zellkern, bevor diese durch einen aktiven Prozess in das Zytoplasma exportiert wird. In Eukaryoten liegen die Informationen für die Proteine fragmentiert vor. Kodierende Sequenzen (Exons) werden unterbrochen von nicht-kodierenden Abschnitten (Introns), welche zunächst beide abgeschrieben werden und die prä-mRNA bilden. Diese initiale RNA-Sequenz muss im Anschluss prozessiert werden, um die Introns zu entfernen und die Exons miteinander zu legieren (Spleißen). Die entstehende neue prä-mRNA wird sofort an ihrem 5‘-Ende methyliert, um sie vor dem Verdau durch 5’-Exonukleasen zu schützen (5‘Capping). Abschließend wird die Transkription terminiert, und um das 3‘-Ende ebenfalls vor einem möglichen Abbau zu schützen, erhalten die Transkripte einen so genannten poly(A)-Schwanz, eine Sequenz aus Adenosinen, die nicht in der DNA-Matrize vorgegeben sind (Polyadenylierung). Diese Prozesse werden durch verschiedene Multi-Protein-RNA-Komplexe im Zusammenspiel mit spezifischen RNA-bindenden Proteinen (RBPs) katalysiert. Das Spleißen wird vom Spliceosom durchgeführt, welches mittels zweier aufeinanderfolgender Umesterungen das Intron zwischen zwei Exons entfernt und die Exons miteinander ligiert. Hierbei können auch ein oder mehrere Exons übersprungen werden. Dieses alternative Spleißen (AS) ermöglicht die Expression alternativer Protein-Isoformen aus demselben Gen. Zusätzlich können durch AS aber auch alternative, toxische Exons in die reife mRNA integriert werden, welche die Stabilität des Transkripts negativ beeinflussen und somit eine Möglichkeit zur Regulation der Proteinexpression bieten. Die Assemblierung des Spliceosoms an der prä-mRNA wird durch die Präsenz von RNA-bindenden Spleiß-Aktivatoren oder -Inhibitoren beeinflusst. Eine bekannte Familie von Spleiß-Aktivatoren ist die der Serin/Arginin-reichen Proteine (SR-Proteine). Diese binden spezifische Sequenzen in Exons und fördern die Assemblierung des Spliceosoms an den jeweiligen Spleißstellen und somit die Inklusion der gebundenen Exons. Dem entgegen wirken Inhibitoren, wie die Proteine aus der hnRNP-Familie, die vorzugsweise in Introns binden. Die Transkription einer neuen prä-mRNA wird durch eine hydrolytische Spaltung in der 3‘-untranslatierten Region (UTR) beendet und das neu entstandene 3‘-Ende dieser prä-mRNA wird durch die neue Synthese eines poly(A)-Schwanzes vor der vorzeitigen Degradation geschützt. Diese zusammenhängenden Prozesse werden von vier Multi-Protein-Komplexen (CFIm, CFIIm, CPSF und CsTF) und der Poly(A)-Polymerase (PAP) katalysiert. Die Adenosin-reiche Sequenz wird durch die Bindung des Poly(A)-bindenden Proteins (PABPN1) stabilisiert wodurch die Aktivität von PAP weiter stimuliert wird. Wie Spleißen ist auch die endonukleolytische Spaltung und Polyadenylierung sequenzspezifisch und abhängig von RBPs, die diese Sequenzen erkennen. Das zentrale Erkennungsmotiv ist das Hexamer ‚AAUAAA‘ sowie bestimmte Varianten dieses Motivs. Dieses so genannte Poly(A)-Signal wird durch die spezifischen Untereinheiten WDR33 und CPSF30 des CPSF-Komplexes erkannt. Die Assemblierung der gesamten Polyadenylierungsmaschinerie wird unterstützt durch den CFIm-Komplex, der UGUA-Motive oberhalb des Poly(A)-Signals bindet sowie durch den CsTF-Komplex, der U/GU-reiche Sequenzen unterhalb des Poly(A)-Signals bindet. Analog zum Spleißen ist auch die Polyadenylierung in den meisten eukaryotischen Genen (bei humanen/murinen Zellen in bis zu 70% der Gene) an mehreren Positionen möglich (alternative Polyadenylierung, APA). Abhängig von der Position der alternativen Polyadenylierungsstellen entstehen dadurch entweder Transkripte mit alternativen terminalen Exons, falls diese Stelle in einem Intron liegt (CDS-APA), oder Transkripte mit unterschiedlich langen 3’UTRs aber identischer kodierender Sequenz, wenn die alternativen Poly(A)-Signale in der 3’UTR liegen (3’UTR-APA). In Abhängigkeit von der Entfernung zum vorherigen STOP-Codon wird die erste Polyadenylierungsstelle (PAS) als ‚proximal‘ (pPAS) und die am weitesten entfernte als ‚distal‘ (dPAS) betitelt. Die Länge der 3’UTR hat Auswirkungen auf die Stabilität, Exporteffizienz, subzelluläre Lokalisation, Translationsrate und lokale Translation der entsprechenden mRNA-Isoform. Einzelne Polyadenylierungsfaktoren wurden mit der Expression bestimmter APA-Isoformen in Verbindung gebracht. Die Reduktion von CFIm führte zur vermehrten Expression von Transkripten mit verkürzten 3‘UTRs, wohingegen verringerte Expressionen von CsTF-Komponenten und von FIP1 (Untereinheit des CPSF-Komplexes) die Expression von Transkripten mit langen 3’UTRs förderte. Bisher sind die Komponenten und Funktionen der einzelnen Polyadenylierungsfaktoren umfassend erforscht, dennoch ist die Regulation der alternativen Polyadenylierung – die Entscheidung, ob die proximale oder distale PAS benutzt wird – weniger entschlüsselt und benötigt zusätzliche Studien. ...

Lifelong Reduction in LDL Cholesterol Due to a Gain-of-Function Mutation in LDLR

Background - Loss-of-function mutations in the LDL receptor gene ( LDLR ) cause elevated levels of LDL cholesterol and premature cardiovascular disease. To date, a gain-of-function mutation in LDLR with a large effect on LDL cholesterol levels has not been described. Here, we searched for sequence variants in LDLR that have a large effect on LDL cholesterol levels. Methods - We analyzed whole-genome sequence (WGS) data from 43,202 Icelanders. Single-nucleotide polymorphisms and structural variants including deletions, insertions and duplications were genotyped using WGS-based data. LDL cholesterol associations were carried out in a sample of >100,000 Icelanders with genetic information (imputed or WGS). Molecular analyses were performed using RNA sequencing and protein expression assays in Epstein-Barr virus-transformed lymphocytes. Results - We discovered a 2.5 kb deletion (del2.5) overlapping the 3' untranslated region (UTR) of LDLR in seven heterozygous carriers from a single family. Mean level of LDL cholesterol was 74% lower in del2.5 carriers than in 101,851 non-carriers, a difference of 2.48 mmol/L (96 mg/dL) ( P =8.4×10 -8 ). Del2.5 results in production of an alternative mRNA isoform with a truncated 3' UTR. The truncation leads to a loss of target sites for microRNAs known to repress translation of LDLR . In Epstein-Barr virus-transformed lymphocytes derived from del2.5 carriers, expression of alternative mRNA isoform was 1.84-fold higher than the wild-type isoform ( P =0.0013) and there was 1.79-fold higher surface expression of the LDL receptor than in non-carriers ( P =0.0086). We did not find a highly penetrant detrimental impact of lifelong very low levels of LDL cholesterol due to del2.5 on health of the carriers. Conclusions - Del2.5 is the first reported gain-of-function mutation in LDLR causing a large reduction in LDL cholesterol. These data point to a role for alternative polyadenylation of LDLR mRNA as a potent regulator of LDL receptor expression in humans.