The RNA encoding the microtubule-associated protein tau has extensive structure that affects its biology

Abstract

Tauopathies are neurodegenerative diseases that affect millions of people worldwide including those with Alzheimer's disease. While many efforts have focused on understanding the role of tau protein in neurodegeneration, there has been little done to systematically analyze and study the structures within tau's encoding RNA and their connection to disease pathology. Knowledge of RNA structure can provide insights into disease mechanisms and how to affect protein production for therapeutic benefit. Using computational methods based on thermodynamic stability and evolutionary conservation, we identified structures throughout the tau pre-mRNA, especially at exonintron junctions and within the 5′ and 3′ untranslated regions (UTRs). In particular, structures were identified at twenty exon-intron junctions. The 5′ UTR contains one structured region, which lies within a known internal ribosome entry site. The 3′ UTR contains eight structured regions, including one that contains a polyadenylation signal.A series of functional experiments were carried out to assess the effects of mutations associated with mis-regulation of alternative splicing of exon 10 and to identify regions of the 3′ UTR that contain cis-regulatory elements. These studies defined novel structural regions within the mRNA that affect stability and pre-mRNA splicing and may lead to new therapeutic targets for treating tau-associated diseases. ABBREVIATIONSAD, Alzheimer's disease; APA, alternative polyadenylation; APSI, average pairwise sequence identity; bp, base pair; DMEM, Dulbecco's Modified Eagle Medium; ED, ensemble diversity; FTDP-17, frontotemporal dementia with parkinsonism-17; htra2β1, transformer 2 beta homolog 1; IRES, internal ribosome entry site; ITAF, IRES trans-acting factor; MAFFT, Multiple Alignment using Fast Fourier Transform; MAPT, microtubule associated protein tau; MBD, microtubule binding domain; MFE, minimum free energy; miRNA, microRNA; mRNA, messenger RNA; mTOR, mammalian target of rapamycin; MSTD, multiple system tauopathy with presenile dementia; ncRNA, noncoding RNA; nt, nucleotide; PD, Parkinson's disease; RNP, ribonucleoprotein;rRNA, ribosomal RNA; SCI, structure conservation index; SD, standard deviation; SF2, serine and arginine rich splicing factor 1, also known as SRSF1; SHAPE, selective 2′ hydroxyl acylation analyzed by primer extension; snRNA, small nuclear RNA; SRp30c, serine and arginine rich splicing factor 9, also known as SRSF9; SRp40, serine and arginine rich splicing factor 5, also known as SRSF5; SRp54, signal recognition particle 54; SRp55, serine and arginine rich splicing factor 6, also known as SRSF6; SVM, support vector machine; UTR, untranslated region

Intrinsic Regulatory Role of RNA Structural Arrangement in Alternative Splicing Control

“…Various mutations in the vicinity of 5′ss of exon 10 increase U1 snRNP binding leading to enhanced production of four-MBDs containing tau proteins [ 280 , 284 ]. Successive discoveries indicated a natural regulatory RNA stem–loop structure embedding 5′ss which due to disease SNVs undergo U1 snRNP-favored destabilization [ 283 , 284 , 285 ].…”

Quantitative prediction of variant effects on alternative splicing using endogenous pre-messenger RNA structure probing

“…Although the structure of the MAPT pre-mRNA was previously studied computationally and in vitro (Varani et al 1999;Lisowiec et al 2015;Tan et al 2019;Chen et al 2019), the structures of the mature 3R and 4R isoforms and MAPT pre-mRNA have not been assessed in their endogenous in vivo context. We used dimethyl sulfate (DMS) to chemically probe RNA structure in T47D and neuronal SH-SY5Y cells and primeramplified the Exon 9-Exon 11 and Exon 9-Exon 10-Exon 11 junctions during library preparation for Mutational-Profiling (-MaP) (Figure 1B; Materials and methods).…”

“…Although NMR, in vitro chemical probing, and computation confirmed the presence of the hairpin (Varani et al 1999;Chen et al 2019;Lisowiec et al 2015), recent studies showed that most RNAs were less structured in vivo and in the nucleus compared with in vitro conditions (Sun et al 2019;Rouskin et al 2014). However, our results revealed that this is not the case for the Exon10-Intron10 junction: in vivo chemical probing of the endogenous junction showcased strong evidence of structure.…”

Advances and Challenges in Understanding MicroRNA Function in Tauopathies: A Case Study of miR-132/212

“…The longest isoform is conserved and highly expressed in the brain (frontal cortex) ( 12 ). Previous studies have shown that different domains within the tau 3′UTR are important for mRNA structure, stability, and transport ( 13 – 15 ). Since tau is a dose-sensitive gene candidate ( 16 , 17 ), and that its mis-regulation is associated with disease ( 18 ), it seems logical that different regulatory mechanisms have evolved to keep tau expression levels in check.…”