RNA-sequencing of a mouse-model of spinal muscular atrophy reveals tissue-wide changes in splicing of U12-dependent introns

Spinal Muscular Atrophy (SMA) is a neuromuscular disorder caused by insufficient levels of the Survival of Motor Neuron (SMN) protein. SMN is expressed ubiquitously and functions in RNA processing pathways that include trafficking of mRNA and assembly of snRNP complexes. Importantly, SMA severity is correlated with decreased snRNP assembly activity. In particular, the minor spliceosomal snRNPs are affected, and some U12-dependent introns have been reported to be aberrantly spliced in patient cells and animal models. SMA is characterized by loss of motor neurons, but the underlying mechanism is largely unknown. It is likely that aberrant splicing of genes expressed in motor neurons is involved in SMA pathogenesis, but increasing evidence indicates that pathologies also exist in other tissues. We present here a comprehensive RNA-seq study that covers multiple tissues in an SMA mouse model. We show elevated U12-intron retention in all examined tissues from SMA mice, and that U12-dependent intron retention is induced upon siRNA knock-down of SMN in HeLa cells. Furthermore, we show that retention of U12-dependent introns is mitigated by ASO treatment of SMA mice and that many transcriptional changes are reversed. Finally, we report on missplicing of several Ca2+ channel genes that may explain disrupted Ca2+ homeostasis in SMA and activation of Cdk5

ANCA patients have T cells responsive to complementary PR-3 antigen

Some patients with proteinase 3 specific anti-neutrophil cytoplasmic autoantibodies (PR3-ANCA) also have antibodies that react to complementary-PR3 (cPR3), a protein encoded by the antisense RNA of the PR3 gene. To study whether patients with anti-cPR3 antibodies have cPR3-responsive memory T cells we selected conditions that allowed cultivation of memory cells but not naïve cells. About half of the patients were found to have CD4+TH1 memory cells responsive to the cPR3138-169-peptide; while only a third of the patients had HI-PR3 protein responsive T cells. A significant number of T cells from patients responded to cPR3138-169 peptide and to HI-PR3 protein by proliferation and/or secretion of IFN-γ, compared to healthy controls while there was no response to scrambled peptide. Cells responsive to cPR3138-169-peptide were not detected in MPO-ANCA patients suggesting that this response is specific. The HLADRB1* 15 allele was significantly overrepresented in our patient group and is predicted to bind cPR3138-169 peptide with high affinity. Regression analysis showed a significant likelihood that anti-cPR3 antibodies and cPR3-specific T cells coexist in individuals, consistent with an immunological history of encounter with a PR3-complementary protein. We suggest that the presence of cells reacting to potential complementary protein pairs might provide an alternative mechanism for auto-immune diseases

Cloned transcription factor MTF-1 activates the mouse metallothionein I promoter

Metallothioneins (MTs) are small cysteine-rich proteins whose structure is conserved from fungi to man. MTs strongly bind heavy metals, notably zinc, copper and cadmium. Upon exposure of cells to heavy metal and other adverse treatments, MT gene transcription is strongly enhanced. Metal induction is mediated by several copies of a 15 bp consensus sequence (metal-responsive element, MRE) present in the promoter region of MT genes. We and others have demonstrated the presence of an MRE-binding factor in HeLa cell nuclear extracts. We found that this factor, termed MTF-1 (MRE-binding transcription factor) is inactivated/reactivated in vitro by zinc withdrawal/addition. Here we report that the amounts of MTF-1-DNA complexes are elevated several-fold in zinc-treated cells, as measured by bandshift assay. We have also cloned the cDNA of mouse MTF-1, a 72.5 kDa protein. MTF-1 contains six zinc fingers and separate transcriptional activation domains with high contents of acidic and proline residues. Ectopic expression of MTF-1 in primate or rodent cells strongly enhances transcription of a reporter gene that is driven by four consensus MREd sites, or by the complete mouse MT-I promoter, even at normal zinc levels

Cloned transcription factor MTF-1 activates the mouse metallothionein I promoter

Metallothioneins (MTs) are small cysteine-rich proteins whose structure is conserved from fungi to man. MTs strongly bind heavy metals, notably zinc, copper and cadmium. Upon exposure of cells to heavy metal and other adverse treatments, MT gene transcription is strongly enhanced. Metal induction is mediated by several copies of a 15 bp consensus sequence (metal-responsive element, MRE) present in the promoter region of MT genes. We and others have demonstrated the presence of an MRE-binding factor in HeLa cell nuclear extracts. We found that this factor, termed MTF-1 (MRE-binding transcription factor) is inactivated/reactivated in vitro by zinc withdrawal/addition. Here we report that the amounts of MTF-1-DNA complexes are elevated several-fold in zinc-treated cells, as measured by bandshift assay. We have also cloned the cDNA of mouse MTF-1, a 72.5 kDa protein. MTF-1 contains six zinc fingers and separate transcriptional activation domains with high contents of acidic and proline residues. Ectopic expression of MTF-1 in primate or rodent cells strongly enhances transcription of a reporter gene that is driven by four consensus MREd sites, or by the complete mouse MT-I promoter, even at normal zinc levels

Cloned transcription factor MTF-1 activates the mouse metallothionein I promoter

Metallothioneins (MTs) are small cysteine-rich proteins whose structure is conserved from fungi to man. MTs strongly bind heavy metals, notably zinc, copper and cadmium. Upon exposure of cells to heavy metal and other adverse treatments, MT gene transcription is strongly enhanced. Metal induction is mediated by several copies of a 15 bp consensus sequence (metal-responsive element, MRE) present in the promoter region of MT genes. We and others have demonstrated the presence of an MRE-binding factor in HeLa cell nuclear extracts. We found that this factor, termed MTF-1 (MRE-binding transcription factor) is inactivated/reactivated in vitro by zinc withdrawal/addition. Here we report that the amounts of MTF-1-DNA complexes are elevated several-fold in zinc-treated cells, as measured by bandshift assay. We have also cloned the cDNA of mouse MTF-1, a 72.5 kDa protein. MTF-1 contains six zinc fingers and separate transcriptional activation domains with high contents of acidic and proline residues. Ectopic expression of MTF-1 in primate or rodent cells strongly enhances transcription of a reporter gene that is driven by four consensus MREd sites, or by the complete mouse MT-I promoter, even at normal zinc levels