Michael Briese

University of Wuerzburg | JMU

Deposition of neurofibrillary tangles composed of hyperphosphorylated tau in the brain is a pathological hallmark and closely correlates with onset and course of Alzheimer's disease. While tau reduction is being pursued as therapeutic strategy, prolonged lowering of total tau might lead to adverse effects, necessitating the development of more targ...

The neuronal RNA-binding protein Ptbp2 regulates neuronal differentiation by modulating alternative splicing programs in the nucleus. Such programs contribute to axonogenesis by adjusting the levels of protein isoforms involved in axon growth and branching. While its functions in alternative splicing have been described in detail, cytosolic roles o...

Chronic arterial hypertension causes cerebral microvascular dysfunction and doubles dementia risk in aging. However, cognitive health preservation by therapeutic blood pressure lowering alone is limited and depends on disease duration, the degree of irreversible tissue damage and whether microvascular function can be restored. This study aimed to u...

Plastin 3 (PLS3) is an F-actin-bundling protein that has gained attention as a modifier of spinal muscular atrophy (SMA) pathology. SMA is a lethal pediatric neuromuscular disease caused by loss of or mutations in the Survival Motor Neuron 1 (SMN1) gene. Pathophysiological hallmarks are cellular maturation defects of motoneurons prior to degenerati...

The P-TEFb complex promotes transcription elongation by releasing paused RNA polymerase II. P-TEFb itself is known to be inacti-vated through binding to the non-coding RNA 7SK but there is only limited information about mechanisms regulating their association. Here, we show that cells deficient in the RNA-binding protein hnRNP R, a known 7SK intera...

Long-term hypertension doubles all-cause dementia risk in aging by structural and functional alterations of the cerebral microvasculature and its surrounding tissue. However, therapeutical blood pressure lowering by itself is limited in its effectiveness depending on the progression of structural vessel remodeling, microvascular dysfunction and par...

Neurons critically rely on the functions of RNA-binding proteins to maintain their polarity and resistance to neurotoxic stress. HnRNP R has a diverse range of post-transcriptional regulatory functions and is important for neuronal development by regulating axon growth. Hnrnpr pre-mRNA undergoes alternative splicing giving rise to a full-length pro...

The noncoding RNA 7SK is a critical regulator of transcription by adjusting the activity of the kinase complex P‐TEFb. Release of P‐TEFb from 7SK stimulates transcription at many genes by promoting productive elongation. Conversely, P‐TEFb sequestration by 7SK inhibits transcription. Recent studies have shown that 7SK functions are particularly imp...

Gene expression requires tight coordination of the molecular machineries that mediate transcription and splicing. While the interplay between transcription kinetics and spliceosome fidelity has been investigated before, less is known about mechanisms regulating the assembly of the spliceosomal machinery in response to transcription changes. Here, w...

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Abstract Protein inclusions containing the RNA-binding protein TDP-43 are a pathological hallmark of amyotrophic lateral sclerosis and other neurodegenerative disorders. The loss of TDP-43 function that is associated with these inclusions affects post-transcriptional processing of RNAs in multiple ways including pre-mRNA splicing, nucleocytoplasmic...

Inflammation and dysregulation of the immune system are hallmarks of several neurodegenerative diseases. An activated immune response is considered to be the cause of myelin breakdown in demyelinating disorders. In the peripheral nervous system (PNS), myelin can be degraded in an autophagy-dependent manner directly by Schwann cells or by macrophage...

Neurotransmission defects and motoneuron degeneration are hallmarks of spinal muscular atrophy, a monogenetic disease caused by the deficiency of the SMN protein. In the present study, we show that systemic application of R-Roscovitine, a Cav2.1/Cav2.2 channel modifier and a cyclin-dependent kinase 5 (Cdk-5) inhibitor, significantly improved surviv...

Studies of spliceosomal interactions are challenging due to their dynamic nature. Here we used spliceosome iCLIP, which immunoprecipitates SmB along with small nuclear ribonucleoprotein particles and auxiliary RNA binding proteins, to map spliceosome engagement with pre-messenger RNAs in human cell lines. This revealed seven peaks of spliceosomal c...

Studies of spliceosomal interactions are challenging due to their dynamic nature. Here we employed spliceosome iCLIP, which immunoprecipitates SmB along with snRNPs and auxiliary RNA binding proteins (RBPs), to simultaneously map human spliceosome engagement with snRNAs and pre-mRNAs. We identify nine sites on pre-mRNAs that overlap with position-d...

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a fatal motoneuron disorder in children with unknown etiology. The disease is caused by mutations in the IGHMBP2 gene, encoding a Super Family 1 (SF1)-type RNA/DNA helicase. IGHMBP2 is a cytosolic protein that binds to ribosomes and polysomes, suggesting a role in mRNA metabolism....

Significance Neurons are highly polarized cells. RNA-binding proteins contribute to this polarization by generating diverse subcellular transcriptomes. The RNA-binding protein hnRNP R is essential for axon growth in motoneurons. This study reports the RNA interactome for hnRNP R. The main interacting RNA of hnRNP R was the noncoding RNA 7SK. Deplet...

In highly polarized cells such as neurons, most RNA molecules are not randomly distributed but sorted into different compartments. So far, methods to analyze the transcriptome in distinct subcellular compartments are not well established. Here, we first describe the culturing of primary motoneurons in compartmentalized chambers to separate the axon...

Most RNAs within polarized cells such as neurons are sorted subcellularly in a coordinated manner. Despite advances in the development of methods for profiling polyadenylated RNAs from small amounts of input RNA, techniques for profiling coding and non-coding RNAs simultaneously are not well established. Here, we optimized a transcriptome profiling...

It is generally believed that splicing removes introns as single units from precursor messenger RNA transcripts. However, some long Drosophila melanogaster introns contain a cryptic site, known as a recursive splice site (RS-site), that enables a multi-step process of intron removal termed recursive splicing. The extent to which recursive splicing...

Neuronal function critically depends on coordinated subcellular distribution of mRNAs. Disturbed mRNA processing and axonal transport has been found in spinal muscular atrophy and could be causative for dysfunction and degeneration of motoneurons. Despite the advances made in characterizing the transport mechanisms of several axonal mRNAs, an unbia...

Illustration of dissection of CA1-hippocampus used for RNAseq experiment. As described in the text. (DOCX)

RNAseq workflows and R-scripts. A pdf file that shows an overview of the workflow used for analyzing RNAseq data in the subcellular fractionation experiments, as well as R-script examples used for ANOVA and permutation analyses. (PDF)

CELF4 is not present in synaptosomes. Synaptosomes were isolated from wildtype mouse cortical brain homogenates. Immunoblot using antibody against CELF4 shows robust signal in input (25 µg total protein), a very faint band in membranous material, and no band in the synaptosome fraction. (DOCX)

Normalized RNAseqdata & iCLIP occupancy & ranks. Spreadsheet that shows the RNA sequencing data from the sucrose gradient fractionation and hippocampal dissection experiments, normalized as described in the text, as well as the CELF4 iCLIP occupancy values and relative ranks from the iCLIP-seq experiments. Genes are listed by Ensembl Gene ID. (XLSX...

GO annotation clustering for top vs. bottom 500 targets ranks. Workbook with multiple worksheets that summarizes the first pass at functional annotation clustering of CELF4 iCLIP targets, comparing the top 500 ranking targets with the bottom 500 ranking targets. Genes are listed by the Mouse Genome Database ID as well as by their International Nome...

Synaptosome methods. Text file that describes the methods for synaptosome analysis, shown in Figure S1. (DOC)

iCLIP primers and summaries, qPCR primers. Text file comprised of five small tables showing primers used for iCLIP and qPCR validation studies, as well as summary statistics for iCLIP results. (DOCX)

CELF4 microarray transcript level normalized data and fstats, and qPCR data and summary statistics. Workbook with multiple worksheets that shows normalized microarray data and results of MAANOVA (F statistics, p-values) and the qPCR validation data. Genes are listed by Ensembl Gene ID. (XLSX)

Posthoc analyses of 142 cb vs. np enriched genes from GO models. Workbook with multiple worksheets that shows the results of the GO analysis of Celf4-genotype dependent subcellular gene expression shifts of CELF4 targets. Genes are listed by the Mouse Genome Database ID. (XLSX)

Fstats & p values for interaction anova - whole set. Workbook with multiple worksheets that shows F-statistics and p-values for the interaction ANOVA analysis of subcellular fractionation or dissection RNAseq experiments. These data are for the whole transcriptome set. Genes are listed by Ensembl Gene ID. (XLSX)

Fstats & p values for interaction anova - CA1 only set. Workbook with multiple worksheets that shows F-statistics and p-values for the interaction ANOVA analysis of subcellular fractionation or dissection RNAseq experiments. These data are for the subset of the whole transcriptome filtered for CA1 synaptic transcripts, as described in the text. Gen...

GO annotation clustering of interaction models. Workbook with multiple worksheets that shows functional annotation clustering of the highest 2000 ranking CELF4 iCLIP targets from the Celf4 genotype-dependent interaction ANOVA analyses of subcellular fractionation or dissection RNAseq experiments, comparing the top 50% F-statistics vs. bottom 50% F-...

RNA-binding proteins have emerged as causal agents of complex neurological diseases. Mice deficient for neuronal RNA-binding protein CELF4 have a complex neurological disorder with epilepsy as a prominent feature. Human CELF4 has recently been associated with clinical features similar to those seen in mutant mice. CELF4 is expressed primarily in ex...

Mice deficient for CELF4, a neuronal RNA-binding protein, have a complex seizure disorder that includes both convulsive and non-convulsive seizures and is dependent upon Celf4 gene dosage and mouse strain background. It was previously shown that Celf4 is expressed predominantly in excitatory neurons, and that deficiency results in abnormal excitato...

TDP-43 is a predominantly nuclear RNA-binding protein that forms inclusion bodies in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The mRNA targets of TDP-43 in the human brain and its role in RNA processing are largely unknown. Using individual nucleotide-resolution ultraviolet cross-linking and immunoprecipitat...

A global view of replicate iCLIP and iCLAP experiments for TIA1 and TIAL1. The three individual replicates of iCLIP for TIA1 or TIAL1 together with iCLAP for either protein are shown in BedGraph format in the UCSC hg18 Genome Browser. cDNA counts at crosslink sites on the sense (purple) or anti-sense strand (orange) of the chromosome 20 are shown....

Reproducibility of replicate iCLIP and iCLAP experiments for TIA1 and TIAL1. (A) Fold-enrichment of pentamers in the 21 nt sequence surrounding crosslink sites (−10 nt to +10 nt) are shown for TIA1 and TIAL1 iCLIP. (B) Pentamer z scores at the 21 nt sequence surrounding crosslink sites (−10 nt to +10 nt) are shown for TIA1 and TIAL1 iCLAP. The sequ...

TIA1/TIAL1 regulate intron retention. (A) iCLIP crosslink sites in the silenced intron in PPIA pre-mRNA. The exon is shown by the rectangle, and the alternative intron by the arrowed line. The area surrounding the 5′ splice site is shown at a greater resolution below. (B) 18 intron retention events detected by the microarray were analysed by real-t...

TIA binding causes distal splicing effects. (A) UCSC hg18 Genome Browser views of iCLIP crosslink sites around the two mutually exclusive exons of FYN pre-mRNA. The areas surrounding both 5′ splice sites are shown at a higher resolution. Capillary electrophoresis of RT-PCR from KD samples and its quantification are shown below. (B) UCSC Genome Brow...

RT-PCR validation of splicing events detected by the microarray using QIAxcel. (A) Cassette exons events validated by QIAxcel and their quantification are shown next to the pictures. (B) Validated alternative 5′ splice sites regulated by the TIA proteins. (C) Validated alternative 3′ splice sites regulated by the TIA proteins. (D) Validated alterna...

RT-PCR primers for validation of splicing event predicted by the microarray. (0.08 MB PDF)

iCLIP and iCLAP of TIA1 and TIAL1. (A) Western blot for TIA1 and TIAL1 in HeLa, TIA1 or TIAL1 overexpressed, and TIA1/TIAL1 double KD cells. GAPDH was used as loading control. (B) Western blot with either antibody upon immunoprecipitation using the same antibody. Protein G beads were used for immunoprecipitation. The strong signal at ±130 kDa repre...

TIA1 and TIAL1 crosslink to different parts of chromosomes. (A) Global view of chromosome 6 with TIA1 and TIAL1 iCLIP crosslink sites. The chromosome is shown at the top. The genes are shown below the tracks. The zoom-in view shows the MAPK14 gene. This gene has two mutually exclusive exons, and splicing change was detected by the microarray. The r...

The location of TIA1 and TIAL1 crosslink sites in previously described pre-mRNAs. (A) TIA1 and TIAL1 crosslink sites in FAS pre-mRNA. In the mid panel, exons 5, 6, and 7 are shown, with exon 6 being alternatively spliced. The arrows above the bar graphs show the previously identified TIA binding sites. (B) TIA1 and TIAL1 crosslink sites in MYC pre-...

Information used to map the sequencing results to genome. (0.04 MB PDF)

Enrichment of pentamers surrounding TIA1 and TIAL1 iCLIP and iCLAP crosslink sites. (0.02 MB PDF)

Supplementary methods. A detailed explanation of the iCLIP, iCLAP, z score analysis, identification of significant iCLIP crosslink sites, and RT-PCR analysis is provided. (0.04 MB DOC)

TIA1/TIAL1 siRNA knock-down in HeLa cells. Western blot for TIA1/TIAL1 KD samples. Either TIA1 or TIAL1 was detected in the upper panels and GAPDH in the bottom panel as loading control. (0.19 MB PDF)

Mapping information for iCLIP and iCLAP data. (0.03 MB PDF)

qPCR primers for validation of intron retention event. (0.06 MB PDF)

The regulation of alternative splicing involves interactions between RNA-binding proteins and pre-mRNA positions close to the splice sites. T-cell intracellular antigen 1 (TIA1) and TIA1-like 1 (TIAL1) locally enhance exon inclusion by recruiting U1 snRNP to 5' splice sites. However, effects of TIA proteins on splicing of distal exons have not yet...

UV cross-linking and immunoprecipitation assay (CLIP) can identify direct interaction sites between RNA-binding proteins and RNAs in vivo, and has been used to study several proteins in tissues and cell cultures. The main challenge of the method is to specifically amplify the low amount of isolated RNA. The current protocol is optimised for efficie...

Spinal muscular atrophy is the most common genetic cause of infant mortality and is characterized by degeneration of lower motor neurons leading to muscle wasting. The causative gene has been identified as survival motor neuron (SMN). The invertebrate model organism Caenorhabditis elegans contains smn-1, the ortholog of human SMN. Caenorhabditis el...

The expression pattern of the survival motor neuron (SMN) protein has been investigated immunohistochemically in the human fetal forebrain from 14 to 38 weeks of gestation. Mutations in the SMN gene cause spinal muscular atrophy (SMA), an autosomal recessive disease characterized by degeneration of lower motor neurons in the spinal cord leading to...

Recent advances in genome research and RNA interference (RNAi) technology have accelerated the adoption of genome-wide experimental approaches for determining gene function in the model organism Caenorhabditis elegans. Despite recent successes, the application of RNAi is limited when gene knockdown causes complex phenotypes or embryonic lethality....

The hereditary neurodegenerative disease spinal muscular atrophy (SMA) with childhood onset is one of the most common genetic causes of infant mortality. The disease is characterized by selective loss of spinal cord motor neurons leading to muscle atrophy and is the result of mutations in the survival motor neuron (SMN) gene. The SMN protein has be...

The distribution pattern of Edg-8 immunostaining in the human developing brain has been investigated with special reference to radial glial fibers. At 24 weeks of gestation, fragments of radial glial fibers are Edg-8-positive within the cortical plate and subplate of allocortical areas. These Edg-8-positive fragments often appear enlarged as varico...

Type-II lissencephaly (LIS-II) is a rare developmental disorder characterized by the excessive migration of neurons and glia, which breach the glia limitans and accumulate within the subarachnoid space. The cortical structure is completely disrupted, the meninges are thickened, and the surface of the brain presents with a ‘cobblestone-like’ appeara...

The G-protein-coupled receptor endothelial differentiation gene-2 (Edg-2) is localized to oligodendrocytes in the postnatal mouse and rat brain. Its spatial and temporal expression pattern has been shown to closely parallel the progress of myelination. Here we have investigated immunohistochemically the distribution of this receptor in 10 fetal for...

Oligodendrocyte-specific protein (OSP)/claudin-11, which is the third most abundant CNS myelin protein, is localized to a variant of tight junctions in myelin sheaths. The distribution of this protein was investigated immunohistochemically in 7 human fetal brains (from 24 to 32 weeks of gestation. At 24 weeks of gestation, OSP/claudin-11-immunoreac...

Intrazerebrale Einblutungen stellen eine häufige Ursache von Komplikationen bei Frühgeborenen dar und haben oftmals geringer ausgeprägte kognitive Leistungen beim Kind zur Folge. Üblicherweise führen solche Einblutungen zu einer Schädigung des Ganglienhügels, einer nur im fetalen Gehirn zu beobachtenden Hirnstruktur, die in der Wandung der laterale...

The Minority Game was introduced to show basic properties of competitive systems with limited common information resources. M. Paczuski and K. E. Bassler introduced a Minority Game with personal limited information resources, where each agent knows the past actions of randomly chosen neighbours [M. Paczuski, K.E. Bassler, Self-organized Networks of...