A Major Fraction of Fibronectin Present in the Extracellular Matrix of Tissues Is Plasma-derived

The origin of the fibronectin (FN) found in the extracellular matrix of tissues has not been defined experimentally. Previous studies suggest that there is contribution from both local tissue production and transfer from plasma, but the extent of this phenomenon has not been addressed. We have shown before that engineered mice constitutively expressing extra domain A-containing FN (EDA+FN) have a significant decrease of FN levels in plasma and most tissues. We showed that hepatocytes modified to produce EDA+FN have normal extracellular matrix-FN levels but secrete less soluble FN. When we performed a liver-specific EDA-exon deletion in these animals, FN levels were restored both in plasma and tissues. Therefore, an important fraction of tissue FN, approximately an equal amount of that produced by the tissue itself, is actually plasma-derived, suggesting that plasma is an important source of tissue FN. The present results have potential significance for understanding the contributions of plasma FN, and perhaps other plasma proteins, in the modulation of cellular activities and in the formation of the extracellular matrix of tissues

Decoding a cancer-relevant splicing decision in the RON proto-oncogene using high-throughput mutagenesis

Mutations causing aberrant splicing are frequently implicated in human diseases including cancer. Here, we establish a high-throughput screen of randomly mutated minigenes to decode the cis-regulatory landscape that determines alternative splicing of exon 11 in the proto-oncogene MST1R (RON). Mathematical modelling of splicing kinetics enables us to identify more than 1000 mutations affecting RON exon 11 skipping, which corresponds to the pathological isoform RON Delta 165. Importantly, the effects correlate with RON alternative splicing in cancer patients bearing the same mutations. Moreover, we highlight heterogeneous nuclear ribonucleoprotein H (HNRNPH) as a key regulator of RON splicing in healthy tissues and cancer. Using iCLIP and synergy analysis, we pinpoint the functionally most relevant HNRNPH binding sites and demonstrate how cooperative HNRNPH binding facilitates a splicing switch of RON exon 11. Our results thereby offer insights into splicing regulation and the impact of mutations on alternative splicing in cancer.Institute of Molecular Biology Core Facilities; DFG [ZA 881/2-1, KO 4566/4-1, LE 3473/2-1]; LOEWE program Ubiquitin Networks (Ub-Net) of the State of Hesse (Germany); Deutsche Forschungsgemeinschaft [SFB902 B13]; EMBO [3057]; Fundacao para a Ciencia e a Tecnologia, Portugal (FCT Investigator Starting Grant) [IF/00595/2014]; German Federal Ministry of Research (BMBF; e:bio junior group program) [FKZ: 0316196]; Boehringer Ingelheim Foundation; [INST 47/870-1 FUGG

The RNA-binding protein PTBP1 is necessary for B cell selection in germinal centers.

Antibody affinity maturation occurs in germinal centers (GCs), where B cells cycle between the light zone (LZ) and the dark zone. In the LZ, GC B cells bearing immunoglobulins with the highest affinity for antigen receive positive selection signals from helper T cells, which promotes their rapid proliferation. Here we found that the RNA-binding protein PTBP1 was needed for the progression of GC B cells through late S phase of the cell cycle and for affinity maturation. PTBP1 was required for proper expression of the c-MYC-dependent gene program induced in GC B cells receiving T cell help and directly regulated the alternative splicing and abundance of transcripts that are increased during positive selection to promote proliferation

Exon structure of the collagen-binding domain of human fibronectin.

Fibronectin consists of a series of three internal homology repeats (types I, II, and III [(1983) Proc. Natl. Acad. Sci. USA 80, 137-141]). The type III units are each coded for by two exons with the exception of the alternatively spliced extra domain type III [(1984) EMBO J. 3, 2511-2516]. To investigate the gene organisation of the type I and II repeats, genomic clones covering the collagen-binding domain of human fibronectin have been isolated and characterised. The results show that each of the type I and II homology units in this region corresponds to an exon. Taken together with the previous data concerning the type III units, the data lend support to a gene fusion model of fibronectin evolution

Genetics and molecular biology

CURR OPIN LIPIDO

The primary structure of the human epsilon-globin gene.

We describe the complete nucleotide sequence of the human epsilon-globin gene including 387 nucleotides of 5' flanking sequence and 301 nucleotides of 3' flanking sequence. The arrangement of coding, noncoding and intervening sequences in this gene is entirely consistent with its identification as the embryonic beta-like globin gene

hnRNP H binding at the 5' splice site correlates with the pathological effect of two intronic mutations in the NF-1 and TSHbeta genes

We have recently reported a disease-causing substitution (+5G > C) at the donor site of NF-1 exon 3 that produces its skipping. We have now studied in detail the splicing mechanism involved in analyzing RNA-protein complexes at several 5' splice sites. Characteristic protein patterns were observed by pulldown and band-shift/super-shift analysis. Here, we show that hnRNP H binds specifically to the wild-type GGGgu donor sequence of the NF-1 exon 3. Depletion analyses shows that this protein restricts the accessibility of U1 small nuclear ribonucleoprotein (U1snRNA) to the donor site. In this context, the +5G > C mutation abolishes both U1snRNP base pairing and the 5' splice site (5'ss) function. However, exon recognition in the mutant can be rescued by disrupting the binding of hnRNP H, demonstrating that this protein enhances the effects of the +5G > C substitution. Significantly, a similar situation was found for a second disease-causing +5G > A substitution in the 5'ss of TSHbeta exon 2, which harbors a GGgu donor sequence. Thus, the reason why similar nucleotide substitutions can be either neutral or very disruptive of splicing function can be explained by the presence of specific binding signatures depending on local contexts

Cell type specific trans-acting factors are involved in alternative splicing of human fibronectin pre-mRNA.

ED-A and ED-B are facultative type III homologies of fibronectin, encoded by alternatively spliced exons, described in man and in rat. A hybrid alpha-globin-fibronectin minigene containing the ED-B region from the human gene has been transfected in human cell lines derived from various tissues, in order to study the processing of the generated precursor RNA in the different cell environments. In most tested lines the pre-RNA is alternatively spliced and produces two mature RNAs, with and without the ED-B exon, in different ratios that closely resemble the corresponding endogenous fibronectin RNAs. In a hepatoma cell line, Hep 3B, only one RNA is produced, in which the ED-B exon is absent; the same pattern of splicing is observed in liver. The data show that all the information required to produce accurate and regulated alternative splicing of the ED-B exon is contained in the fragment used and cell specific factors are necessary for the pre-RNA to be differentially spliced in the various cell lines. In contrast, expression in Hep 3B of a similar gene containing the ED-A area failed to reproduce the liver specific splicing pattern. Therefore regulation of ED-A processing is likely to involve different mechanisms to those responsible for control of ED-B splicing