ApoE maintains neuronal integrity via microRNA and H3K27me3-mediated repression.

ApoE regulates neurogenesis, although how it influences genetic programs remains elusive. Cortical neurons induced from isogenic control and ApoE-/- human neural stem cells (NSCs) recapitulated key transcriptomic signatures of in vivo counterparts identified from single-cell human midbrain. Surprisingly, ApoE expression in NSC and neural progenitor cells (NPCs) is not required for differentiation. Instead, ApoE prevents the over-proliferation of non-neuronal cells during extended neuronal culture when it is not expressed. Elevated miR-199a-5p level in ApoE-/- cells lowers the EZH1 protein and the repressive H3K27me3 mark, a phenotype rescued by miR-199a-5p steric inhibitor. Reduced H3K27me3 at genes linked to extracellular matrix organization and angiogenesis in ApoE-/- NPC correlates with their aberrant expression and phenotypes in neurons. Interestingly, the ApoE coding sequence, which contains many predicted miR-199a-5p binding sites, can repress miR-199a-5p without translating into protein. This suggests that ApoE maintains neurons integrity through the target-directed miRNA degradation of miR-199a-5p, imparting the H3K27me3-mediated repression of non-neuronal genes during differentiation.

RNA Secondary Structure-Based Design of Antisense Peptide Nucleic Acids for Modulating Disease-Associated Aberrant Tau Pre-mRNA Alternative Splicing.

Alternative splicing of tau pre-mRNA is regulated by a 5' splice site (5'ss) hairpin present at the exon 10-intron 10 junction. Single mutations within the hairpin sequence alter hairpin structural stability and/or the binding of splicing factors, resulting in disease-causing aberrant splicing of exon 10. The hairpin structure contains about seven stably formed base pairs and thus may be suitable for targeting through antisense strands. Here, we used antisense peptide nucleic acids (asPNAs) to probe and target the tau pre-mRNA exon 10 5'ss hairpin structure through strand invasion. We characterized by electrophoretic mobility shift assay the binding of the designed asPNAs to model tau splice site hairpins. The relatively short (10-15 mer) asPNAs showed nanomolar binding to wild-type hairpins as well as a disease-causing mutant hairpin C+19G, albeit with reduced binding strength. Thus, the structural stabilizing effect of C+19G mutation could be revealed by asPNA binding. In addition, our cell culture minigene splicing assay data revealed that application of an asPNA targeting the 3' arm of the hairpin resulted in an increased exon 10 inclusion level for the disease-associated mutant C+19G, probably by exposing the 5'ss as well as inhibiting the binding of protein factors to the intronic spicing silencer. On the contrary, the application of asPNAs targeting the 5' arm of the hairpin caused an increased exon 10 exclusion for a disease-associated mutant C+14U, mainly by blocking the 5'ss. PNAs could enter cells through conjugation with amino sugar neamine or by cotransfection with minigene plasmids using a commercially available transfection reagent.

A Disease-Causing Intronic Point Mutation C19G Alters Tau Exon 10 Splicing via RNA Secondary Structure Rearrangement.

Alternative splicing of MAPT cassette exon 10 produces tau isoforms with four microtubule-binding repeat domains (4R) upon exon inclusion or three repeats (3R) upon exon skipping. In human neurons, deviations from the ∼1:1 physiological 4R:3R ratio lead to frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). Certain FTDP-17-associated mutations affect a regulatory hairpin that sequesters the exon 10 5' splice site (5'ss, located at the exon 10-intron 10 junction). These mutations tend to increase the 4R:3R ratio by destabilizing the hairpin, thereby improving 5'ss recognition by U1 snRNP. Interestingly, a single C-to-G mutation at the 19th nucleotide in intron 10 (C19G or +19G) decreases the level of exon 10 inclusion significantly from 56% to 1%, despite the disruption of a G-C base pair in the bottom stem of the hairpin. Here, we show by biophysical characterization, including thermal melting, fluorescence, and single-molecule mechanical unfolding using optical tweezers, that the +19G mutation alters the structure of the bottom stem, resulting in the formation of a new bottom stem with enhanced stability. The cell culture alternative splicing patterns of a series of minigenes reveal that the splicing activities of the mutants with destabilizing mutations on the top stem can be compensated in a position-dependent manner by the +19G mutation in the bottom stem. We observed an excellent correlation between the level of exon 10 inclusion and the rate of mechanical unfolding at 10 pN, indicating that the unfolding of the splice site hairpins (to facilitate subsequent binding of U1 snRNA) may be aided by helicases or other proteins.

Noncanonical registers and base pairs in human 5' splice-site selection.

Accurate recognition of splice sites is essential for pre-messenger RNA splicing. Mammalian 5' splice sites are mainly recognized by canonical base-pairing to the 5' end of U1 small nuclear RNA, yet we described multiple noncanonical base-pairing registers by shifting base-pair positions or allowing one-nucleotide bulges. By systematic mutational and suppressor U1 analyses, we prove three registers involving asymmetric loops and show that two-nucleotide bulges but not longer can form in this context. Importantly, we established that a noncanonical uridine-pseudouridine interaction in the 5' splice site/U1 helix contributes to the recognition of certain 5' splice sites. Thermal melting experiments support the formation of noncanonical registers and uridine-pseudouridine interactions. Overall, we experimentally validated or discarded the majority of predicted noncanonical registers, to derive a list of 5' splice sites using such alternative mechanisms that is much different from the original. This study allows not only the mechanistic understanding of the recognition of a wide diversity of mammalian 5' splice sites, but also the future development of better splice-site scoring methods that reliably predict the effects of disease-causing mutations at these sequences.

Informational Suppression to Probe RNA:RNA Interactions in the Context of Ribonucleoproteins: U1 and 5' Splice-Site Base-Pairing.

Informational suppression is a method to map specific RNA:RNA interactions by taking advantage of the rules of base complementarity. First, a predicted Watson-Crick base pair is broken by single-nucleotide substitution which disrupts the RNA's structure and/or function. Second, the base pair is restored by mutating the opposing nucleotide, thereby rescuing structure and/or function. This method applies to RNP:RNA interactions such as 5' splice-site (5'ss) base-pairing to the 5' end of U1 small nuclear RNA as part of a small nuclear RNP. Our protocol aims to determine the 5'ss:U1 base-pairing register for natural 5'ss, because for distinct 5'ss sequences the nucleotides on each strand can be aligned differently. This methodology includes cloning of a wild-type splicing minigene and introduction of 5'ss variants by PCR mutagenesis. A U1-expression plasmid is mutated to construct "suppressor U1" snRNAs with restored base-pairing to mutant 5'ss in different registers. Cells are transfected with combinations of minigenes and suppressor U1s, and the splicing patterns are analyzed by reverse transcription and semiquantitative PCR, followed by gel electrophoresis. The identity of suppressor U1s that rescue splicing for specific mutations indicates the register used in that 5'ss. We also provide tips to adapt this protocol to other minigenes or registers.