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Correct pre-mRNA processing in higher eukaryotes vastly depends on splice site recognition. Beyond conserved 5′ss and 3′ss motifs, splicing regulatory elements (SREs) play a pivotal role in this recognition process. Here, we present in silico designed sequences with arbitrary a priori prescribed splicing regulatory HEXplorer properties that can be concatenated to arbitrary length without changing their regulatory properties. We experimentally validated in silico predictions in a massively parallel splicing reporter assay on more than 3000 sequences and exemplarily identified some SRE binding proteins. Aiming at a unified ‘functional splice site strength’ encompassing both U1 snRNA complementarity and impact from neighboring SREs, we developed a novel RNA-seq based 5′ss usage landscape, mapping the competition of pairs of high confidence 5′ss and neighboring exonic GT sites along HBond and HEXplorer score coordinate axes on human fibroblast and endothelium transcriptome datasets. These RNA-seq data served as basis for a logistic 5′ss usage prediction model, which greatly improved discrimination between strong but unused exonic GT sites and annotated highly used 5′ss. Our 5′ss usage landscape offers a unified view on 5′ss and SRE neighborhood impact on splice site recognition, and may contribute to improved mutation assessment in human genetics.

Biallelic germline mutations in BRCA2 occur in the Fanconi anemia (FA)-D1 subtype of the rare pediatric disorder, FA, characterized clinically by severe congenital abnormalities and a very high propensity to develop malignancies early in life. Clinical and genetic data from 96 FA-D1 patients with biallelic BRCA2 mutations were collected and used to develop a new cancer risk prediction score system based on the specific mutations in BRCA2. This score takes into account the location of frameshift/stop and missense mutations relative to exon 11 of BRCA2, which encodes the major sites for interaction with the RAD51 recombinase, and uses the MaxEnt and HBond splicing scores to analyze potential splice site perturbations. Among 75 FA-D1 patients with ascertained BRCA2 mutations, 66 patients developed 102 malignancies, ranging from one to three independent tumors per individual. The median age at the clinical presentation of peripheral embryonal tumors was 1.0, at the onset of hematologic malignancies 1.8 and at the manifestation of CNS tumors 2.7 years, respectively. Patients who received treatment lived longer than those without. Using our novel scoring system, we could distinguish three distinct cancer risk groups among FA-D1 patients: in the first, patients developed their initial malignancy at a median age of 1.3 years (n = 36, 95% CI = 0.9–1.8), in the second group at 2.3 years (n = 17, 95% CI = 1.4–4.4) and in the third group at 23.0 years (n = 22, 95% CI = 4.3—n/a). Therefore, this scoring system allows, for the first time, to predict the cancer manifestation of FA-D1 patients simply based on the type and position of the mutations in BRCA2.

Background The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has led to the development of various vaccines. Real-life data on immune responses elicited in the most vulnerable group of vaccinees older than age 80 years old are still underrepresented despite the prioritization of the elderly in vaccination campaigns. Methods We conducted a cohort study with 2 age groups, young vaccinees below the age of 60 years and elderly vaccinees over the age of 80 years, to compare their antibody responses to the first and second dose of the BNT162b2 coronavirus disease 2019 vaccination. Results Although the majority of participants in both groups produced specific immunoglobulin G antibody titers against SARS-CoV-2 spike protein, titers were significantly lower in elderly participants. Although the increment of antibody levels after the second immunization was higher in elderly participants, the absolute mean titer of this group remained lower than the Conclusions Our data showed differences between the antibody responses raised after the first and second BNT162b2 vaccination, in particular lower frequencies of neutralizing antibodies in the elderly group. This suggests that this population needs to be closely monitored and may require earlier revaccination and/or an increased vaccine dose to ensure stronger long-lasting immunity and protection against infection.

After human immunodeficiency virus type 1 (HIV-1) was identified in the early 1980s, intensive work began to understand the molecular basis of HIV-1 gene expression. Subgenomic HIV-1 RNA regions, spread throughout the viral genome, were described to have a negative impact on the nuclear export of some viral transcripts. Those studies revealed an intrinsic RNA code as a new form of nuclear export regulation. Since such regulatory regions were later also identified in other viruses, as well as in cellular genes, it can be assumed that, during evolution, viruses took advantage of them to achieve more sophisticated replication mechanisms. Here, we review HIV-1 cis-acting repressive sequences that have been identified, and we discuss their possible underlying mechanisms and importance. Additionally, we show how current bioinformatic tools might allow more predictive approaches to identify and investigate them.

After human immunodeficiency virus type 1 (HIV-1) was identified in the early 1980s, intensive work began to understand the molecular basis of HIV-1 gene expression. Subgenomic HIV-1 RNA regions, spread throughout the viral genome, were described to have a negative impact on the nuclear export of some viral transcripts. Those studies revealed an intrinsic RNA code as a new form of nuclear export regulation. Since such regulatory regions were later also identified in other viruses, as well as in cellular genes, it can be assumed that, during evolution, viruses took advantage of them to achieve more sophisticated replication mechanisms. Here, we review HIV-1 cis-acting repressive sequences that have been identified, and we discuss their possible underlying mechanisms and importance. Additionally, we show how current bioinformatic tools might allow more predictive approaches to identify and investigate them.

BackgroundThe SARS-CoV-2 pandemic has led to the development of various vaccines. Real-life data on immune responses elicited in the most vulnerable group of vaccinees over 80 years old is still underrepresented despite the prioritization of the elderly in vaccination campaigns.MethodsWe conducted a cohort study with two age groups, young vaccinees below the age of 60 and elderly vaccinees over the age of 80, to compare their antibody responses to the first and second dose of the BNT162b2 COVID-19 vaccination.ResultsWhile the majority of participants in both groups produced specific IgG antibody titers against SARS-CoV-2 spike protein, titers were significantly lower in elderly participants. Although the increment of antibody levels after the second immunization was higher in elderly participants, the absolute mean titer of this group remained lower than the ConclusionOur data suggests that lower frequencies of neutralizing antibodies after BNT162b2 vaccination in the elderly population may require earlier revaccination to ensure strong immunity and protection against infection.

Glioblastoma multiforme (GBM) possesses glioma stem cells (GSCs) that promote self-renewal, tumor propagation, and relapse. GBM has a poor prognosis, and currently, there are no curative options exist. Understanding the mechanisms of GSCs self-renewal can offer targeted therapeutic interventions. However, insufficient knowledge of the fundamental biology of GSCs is a significant bottleneck hindering these efforts. Here, we show that patient-derived GSCs recruit an elevated level of proteins that ensure the temporal cilium disassembly, leading to suppressed ciliogenesis. Depleting the cilia disassembly complex components at the ciliary base is sufficient to induce ciliogenesis in a subset of GSCs. Importantly, restoring ciliogenesis caused GSCs to behave like healthy NPCs switching from self-renewal to differentiation. Finally, using an organoid-based glioma invasion assay and brain xenografts in mice, we establish that ciliogenesis-induced differentiation can prevent the infiltration of GSCs into the brain. Our findings illustrate a crucial role for cilium as a molecular switch in determining GSCs’ fate and suggest that cilium induction is an attractive strategy to intervene in GSCs proliferation.

Reporting of a single nucleotide variant (SNV) follows the Sequence Variant Nomenclature ( http://varnomen.hgvs.org/ ), using an unambiguous numbering scheme specific for coding and noncoding DNA. However, the corresponding sequence neighborhood of a given SNV, which is required to assess its impact on splicing regulation, is not easily accessible from this nomenclature. Providing fast and easy access to this neighborhood just from a given SNV reference, the novel tool VarCon combines information of the Ensembl human reference genome and the corresponding transcript table for accurate retrieval. VarCon also displays splice site scores (HBond and MaxEnt scores) and HEXplorer profiles of an SNV neighborhood, reflecting position-dependent splice enhancing and silencing properties.

Recently, by combining transcriptomics with functional splicing reporter assays we were able to identify GT > GC > TT as the three highest ranked dinucleotides of human 5ʹ splice sites (5’ss). Here, we have extended our investigations to the proteomic characterization of nuclear proteins that bind to canonical and noncanonical 5’ss. Surprisingly, we found that U1 snRNP binding to functional 5’ss sequences prevented components of the DNA damage response (DDR) from binding to the RNA, suggesting a close link between spliceosome arrangement and genome stability. We demonstrate that all tested noncanonical 5’ss sequences are bona-fide targets of the U2-type spliceosome and are bound by U1 snRNP, including U1-C, in the presence of splicing enhancers. The quantity of precipitated U1-C protein was similar for all noncanonical 5’ss dinucleotides, so that the highly different 5’ss usage was likely due to a later step after early U1 snRNP binding. In addition, we show that an internal GT at positions +5/+6 can be advantageous for splicing at position +1 of noncanonical splice sites. Likewise, and in agreement with previous observations, splicing inactive U1 snRNP binding sites could serve as splicing enhancers, which may also explain the higher abundance of U1 snRNPs compared to other U snRNPs. Finally, we observe that an arginine-serine (RS)-rich domain recruitment to stem loop I of the U1 snRNA is functionally sufficient to promote exon-definition and upstream 3’ss activation.

The underlying molecular mechanism and their general effect on the replication capacity of HIV 1 drug-resistance-associated mutations is often poorly understood. To elucidate the effect of two such mutations located in a region with a high density of spicing regulatory elements on the HIV-1-splicing outcome, bioinformatic predictions were combined with transfection and infection experiments. Results show that the previously described R263K drug-resistance-associated integrase mutation has additionally a severe effect on the ESE2b splicing regulatory element (SRE) in exon 2b, which causes loss of SD2b recognition. This was confirmed by an R263R silent mutation with a similar predicted effect on the exon 2b SRE. In contrast, a V260I mutation and its silent counterpart with a lower effect on ESS2b did not exhibit any differences in the splicing pattern. Since HIV-1 highly relies on a balanced splicing reaction, changes in the splicing outcome can contribute to changes in viral replication and might add to the effect of escape mutations toward antiviral drugs. Thus, a classification of mutations purely addressing proteins is insufficient.

Most human pathogenic mutations in 5′ splice sites affect the canonical GT in positions +1 and +2, leading to noncanonical dinucleotides. On the other hand, noncanonical dinucleotides are observed under physiological conditions in ∼1% of all human 5′ss. It is therefore a challenging task to understand the pathogenic mutation mechanisms underlying the conditions under which noncanonical 5′ss are used. In this work, we systematically examined noncanonical 5′ splice site selection, both experimentally using splicing competition reporters and by analyzing a large RNA-seq data set of 54 fibroblast samples from 27 subjects containing a total of 2.4 billion gapped reads covering 269,375 exon junctions. From both approaches, we consistently derived a noncanonical 5′ss usage ranking GC > TT > AT > GA > GG > CT. In our competition splicing reporter assay, noncanonical splicing was strictly dependent on the presence of upstream or downstream splicing regulatory elements (SREs), and changes in SREs could be compensated by variation of U1 snRNA complementarity in the competing 5′ss. In particular, we could confirm splicing at different positions (i.e., −1, +1, +5) of a splice site for all noncanonical dinucleotides “weaker” than GC. In our comprehensive RNA-seq data set analysis, noncanonical 5′ss were preferentially detected in weakly used exon junctions of highly expressed genes. Among high-confidence splice sites, they were 10-fold overrepresented in clusters with a neighboring, more frequently used 5′ss. Conversely, these more frequently used neighbors contained only the dinucleotides GT, GC, and TT, in accordance with the above ranking.

Genomic alignments of sequenced cellular messenger RNA contain gapped alignments which are interpreted as consequence of intron removal. The resulting gap-sites, genomic locations of alignment gaps, are landmarks representing potential splice-sites. As alignment algorithms report gap-sites with a considerable false discovery rate, validations are required. We describe two quality scores, gap quality score (gqs) and weighted gap information score (wgis), developed for validation of putative splicing events: While gqs solely relies on alignment data wgis additionally considers information from the genomic sequence. FASTQ files obtained from 54 human dermal fibroblast samples were aligned against the human genome (GRCh38) using TopHat and STAR aligner. Statistical properties of gap-sites validated by gqs and wgis were evaluated by their sequence similarity to known exon-intron borders. Within the 54 samples, TopHat identifies 1,000,380 and STAR reports 6,487,577 gap-sites. Due to the lack of strand information, however, the percentage of identified GT-AG gap-sites is rather low. While gap-sites from TopHat contain ≈89% GT-AG, gap-sites from STAR only contain ≈42% GT-AG dinucleotide pairs in merged data from 54 fibroblast samples. Validation with gqs yields 156,251 gap-sites from TopHat alignments and 166,294 from STAR alignments. Validation with wgis yields 770,327 gap-sites from TopHat alignments and 1,065,596 from STAR alignments. Both alignment algorithms, TopHat and STAR, report gap-sites with considerable false discovery rate, which can drastically be reduced by validation with gqs and wgis.

Elaborate research on splicing, starting in the late seventies, evolved from the discovery that 5' splice sites are recognized by their complementarity to U1 snRNA towards the realization that RNA duplex formation cannot be the sole basis for 5'ss selection. Rather, their recognition is highly influenced by a number of context factors including transcript architecture as well as splicing regulatory elements (SREs) in the splice site neighborhood. In particular, proximal binding of splicing regulatory proteins highly influences splicing outcome. The importance of SRE integrity especially becomes evident in the light of human pathogenic mutations where single nucleotide changes in SREs can severely affect the resulting transcripts. Bioinformatics tools nowadays greatly assist in the computational evaluation of 5'ss, their neighborhood and the impact of pathogenic mutations. Although predictions are already quite robust, computational evaluation of the splicing regulatory landscape still faces challenges to increase future reliability. This article is part of a Special Issue entitled: RNA structure and splicing regulation edited by Francisco Baralle, Ravindra Singh and Stefan Stamm.

A critical step in exon definition is the recognition of a proper splice donor (5΄ss) by the 5’ end of U1 snRNA. In the selection of appropriate 5΄ss, cis-acting splicing regulatory elements (SREs) are indispensable. As a model for 5΄ss recognition, we investigated cryptic 5΄ss selection within the human fibrinogen Bβ-chain gene (FGB) exon 7, where we identified several exonic SREs that simultaneously acted on up- and downstream cryptic 5΄ss. In the FGB exon 7 model system, 5΄ss selection iteratively proceeded along an alternating sequence of U1 snRNA binding sites and interleaved SREs which in principle supported different 3’ exon ends. Like in a relay race, SREs either suppressed a potential 5΄ss and passed the splicing baton on or splicing actually occurred. From RNA-Seq data, we systematically selected 19 genes containing exons with silent U1 snRNA binding sites competing with nearby highly used 5΄ss. Extensive SRE analysis by different algorithms found authentic 5΄ss significantly more supported by SREs than silent U1 snRNA binding sites, indicating that our concept may permit generalization to a model for 5΄ss selection and 3’ exon end definition.