Mapping Of Cleavage Sites Research Articles

Mycobacterial RNase E cleaves with a distinct sequence preference and controls the degradation rates of most Mycolicibacterium smegmatis mRNAs

The mechanisms and regulation of RNA degradation in mycobacteria have been subject to increased interest following the identification of interplay between RNA metabolism and drug resistance. Mycobacteria encode multiple ribonucleases predicted to participate in mRNA degradation and/or processing of stable RNAs. RNase E is hypothesized to play a major role in mRNA degradation because of its essentiality in mycobacteria and its role in mRNA degradation in gram-negative bacteria. Here, we defined the impact of RNase E on mRNA degradation rates transcriptome-wide in the nonpathogenic model Mycolicibacterium smegmatis. RNase E played a rate-limiting role in degradation of the transcripts encoded by at least 89% of protein-coding genes, with leadered transcripts often being more affected by RNase E repression than leaderless transcripts. There was an apparent global slowing of transcription in response to knockdown of RNase E, suggesting that M.smegmatis regulates transcription in responses to changes in mRNA degradation. This compensation was incomplete, as the abundance of most transcripts increased upon RNase E knockdown. We assessed the sequence preferences for cleavage by RNase E transcriptome-wide in M.smegmatis and Mycobacterium tuberculosis and found a consistent bias for cleavage in C-rich regions. Purified RNase E had a clear preference for cleavage immediately upstream of cytidines, distinct from the sequence preferences of RNase E in gram-negative bacteria. We furthermore report a high-resolution map of mRNA cleavage sites in M. tuberculosis, which occur primarily within the RNase E-preferred sequence context, confirming that RNase E has a broad impact on the M.tuberculosis transcriptome.

Genome-wide mapping of fluoroquinolone-stabilized DNA gyrase cleavage sites displays drug specific effects that correlate with bacterial persistence.

Bacterial persisters are rare phenotypic variants that are suspected to be culprits of recurrent infections. Fluoroquinolones (FQs) are a class of antibiotics that facilitate bacterial killing by stabilizing bacterial type II topoisomerases when they are in a complex with cleaved DNA. In Escherichia coli, DNA gyrase is the primary FQ target, and previous work has demonstrated that persisters are not spared from FQ-induced DNA damage. Since DNA gyrase cleavage sites (GCSs) largely govern the sites of DNA damage from FQ treatment, we hypothesized that GCS characteristics (e.g. number, strength, location) may influence persistence. To test this hypothesis, we measured genome-wide GCS distributions after treatment with a panel of FQs in stationary-phase cultures. We found drug-specific effects on the GCS distribution and discovered a strong negative correlation between the genomic cleavage strength and FQ persister levels. Further experiments and analyses suggested that persistence was unlikely to be governed by cleavage to individual sites, but rather survival was a function of the genomic GCS distribution. Together, these findings demonstrate FQ-specific differences in GCS distribution that correlate with persister levels and suggest that FQs that better stabilize DNA gyrase in cleaved complexes with DNA will lead to lower levels of persistence.

Transcriptome-wide in vivo mapping of cleavage sites for the compact cyanobacterial ribonuclease E reveals insights into its function and substrate recognition.

Ribonucleases are crucial enzymes in RNA metabolism and post-transcriptional regulatory processes in bacteria. Cyanobacteria encode the two essential ribonucleases RNase E and RNase J. Cyanobacterial RNase E is shorter than homologues in other groups of bacteria and lacks both the chloroplast-specific N-terminal extension as well as the C-terminal domain typical for RNase E of enterobacteria. In order to investigate the function of RNase E in the model cyanobacterium Synechocystis sp. PCC 6803, we engineered a temperature-sensitive RNase E mutant by introducing two site-specific mutations, I65F and the spontaneously occurred V94A. This enabled us to perform RNA-seq after the transient inactivation of RNase E by a temperature shift (TIER-seq) and to map 1472 RNase-E-dependent cleavage sites. We inferred a dominating cleavage signature consisting of an adenine at the −3 and a uridine at the +2 position within a single-stranded segment of the RNA. The data identified mRNAs likely regulated jointly by RNase E and an sRNA and potential 3′ end-derived sRNAs. Our findings substantiate the pivotal role of RNase E in post-transcriptional regulation and suggest the redundant or concerted action of RNase E and RNase J in cyanobacteria.

RNA Sequencing for Transcript 5'-End Mapping in Mycobacteria.

Next-generation sequencing technologies facilitate the analysis of multiple important properties of transcriptomes in addition to gene expression levels. Here, we describe a method for mapping RNA 5' ends in Mycobacterium tuberculosis and Mycobacterium smegmatis, which allows the determination of transcription start sites (TSSs), comparative analysis of promoter usage under different conditions, and mapping of endoribonucleolytic cleavage sites. We describe in detail the procedures for constructing RNA sequencing libraries appropriate for RNA 5' end mapping using an Illumina sequencing platform, as well as bioinformatic procedures for data analysis.

Defining the Transcriptional and Post-transcriptional Landscapes of Mycobacterium smegmatis in Aerobic Growth and Hypoxia.

The ability of Mycobacterium tuberculosis to infect, proliferate, and survive during long periods in the human lungs largely depends on the rigorous control of gene expression. Transcriptome-wide analyses are key to understanding gene regulation on a global scale. Here, we combine 5′-end-directed libraries with RNAseq expression libraries to gain insight into the transcriptome organization and post-transcriptional mRNA cleavage landscape in mycobacteria during log phase growth and under hypoxia, a physiologically relevant stress condition. Using the model organism Mycobacterium smegmatis, we identified 6,090 transcription start sites (TSSs) with high confidence during log phase growth, of which 67% were categorized as primary TSSs for annotated genes, and the remaining were classified as internal, antisense, or orphan, according to their genomic context. Interestingly, over 25% of the RNA transcripts lack a leader sequence, and of the coding sequences that do have leaders, 53% lack a strong consensus Shine-Dalgarno site. This indicates that like M. tuberculosis, M. smegmatis can initiate translation through multiple mechanisms. Our approach also allowed us to identify over 3,000 RNA cleavage sites, which occur at a novel sequence motif. To our knowledge, this represents the first report of a transcriptome-wide RNA cleavage site map in mycobacteria. The cleavage sites show a positional bias toward mRNA regulatory regions, highlighting the importance of post-transcriptional regulation in gene expression. We show that in low oxygen, a condition associated with the host environment during infection, mycobacteria change their transcriptomic profiles and endonucleolytic RNA cleavage is markedly reduced, suggesting a mechanistic explanation for previous reports of increased mRNA half-lives in response to stress. In addition, a number of TSSs were triggered in hypoxia, 56 of which contain the binding motif for the sigma factor SigF in their promoter regions. This suggests that SigF makes direct contributions to transcriptomic remodeling in hypoxia-challenged mycobacteria. Taken together, our data provide a foundation for further study of both transcriptional and posttranscriptional regulation in mycobacteria.

Single-nucleotide-resolution mapping of DNA gyrase cleavage sites across the Escherichia coli genome.

An important antibiotic target, DNA gyrase is an essential bacterial enzyme that introduces negative supercoils into DNA and relaxes positive supercoils accumulating in front of moving DNA and RNA polymerases. By altering the superhelical density, gyrase may regulate expression of bacterial genes. The information about how gyrase is distributed along genomic DNA and whether its distribution is affected by drugs is scarce. During catalysis, gyrase cleaves both DNA strands forming a covalently bound intermediate. By exploiting the ability of several topoisomerase poisons to stabilize this intermediate we developed a ChIP-Seq-based approach to locate, with single nucleotide resolution, DNA gyrase cleavage sites (GCSs) throughout the Escherichia coli genome. We identified an extended gyrase binding motif with phased 10-bp G/C content variation, indicating that bending ability of DNA contributes to gyrase binding. We also found that GCSs are enriched in extended regions located downstream of highly transcribed operons. Transcription inhibition leads to redistribution of gyrase suggesting that the enrichment is functionally significant. Our method can be applied for precise mapping of prokaryotic and eukaryotic type II topoisomerases cleavage sites in a variety of organisms and paves the way for future studies of various topoisomerase inhibitors.

Single-nucleotide-resolution mapping of DNA gyrase cleavage sites across the Escherichia coli genome.

An important antibiotic target, DNA gyrase is an essential bacterial enzyme that introduces negative supercoils into DNA and relaxes positive supercoils accumulating in front of moving DNA and RNA polymerases. By altering the superhelical density, gyrase may regulate expression of bacterial genes. The information about how gyrase is distributed along genomic DNA and whether its distribution is affected by drugs is scarce. During catalysis, gyrase cleaves both DNA strands forming a covalently bound intermediate. By exploiting the ability of several topoisomerase poisons to stabilize this intermediate we developed a ChIP-Seq-based approach to locate, with single nucleotide resolution, DNA gyrase cleavage sites (GCSs) throughout the Escherichia coli genome. We identified an extended gyrase binding motif with phased 10-bp G/C content variation, indicating that bending ability of DNA contributes to gyrase binding. We also found that GCSs are enriched in extended regions located downstream of highly transcribed operons. Transcription inhibition leads to redistribution of gyrase suggesting that the enrichment is functionally significant. Our method can be applied for precise mapping of prokaryotic and eukaryotic type II topoisomerases cleavage sites in a variety of organisms and paves the way for future studies of various topoisomerase inhibitors.

Caspase Cleavage of Gelsolin Is an Inductive Cue for Pathologic Cardiac Hypertrophy.

BackgroundCardiac hypertrophy is an adaptive remodeling event that may improve or diminish contractile performance of the heart. Physiologic and pathologic hypertrophy yield distinct outcomes, yet both are dependent on caspase‐directed proteolysis. This suggests that each form of myocardial growth may derive from a specific caspase cleavage event(s). We examined whether caspase 3 cleavage of the actin capping/severing protein gelsolin is essential for the development of pathologic hypertrophy.Methods and ResultsCaspase targeting of gelsolin was established through protein analysis of hypertrophic cardiomyocytes and mass spectrometry mapping of cleavage sites. Pathologic agonists induced late‐stage caspase‐mediated cleavage of gelsolin. The requirement of caspase‐mediated gelsolin cleavage for hypertrophy induction was evaluated in primary cardiomyocytes by cell size analysis, monitoring of prohypertrophy markers, and measurement of hypertrophy‐related transcription activity. The in vivo impact of caspase‐mediated cleavage was investigated by echo‐guided intramyocardial injection of adenoviral‐expressed gelsolin. Expression of the N‐terminal gelsolin caspase cleavage fragment was necessary and sufficient to cause pathologic remodeling in isolated cardiomyocytes and the intact heart, whereas expression of a noncleavable form prevents cardiac remodeling. Alterations in myocardium structure and function were determined by echocardiography and end‐stage cardiomyocyte cell size analysis. Gelsolin secretion was also monitored for its impact on naïve cells using competitive antibody trapping, demonstrating that hypertrophic agonist stimulation of cardiomyocytes leads to gelsolin secretion, which induces hypertrophy in naïve cells.ConclusionsThese results suggest that cell autonomous caspase cleavage of gelsolin is essential for pathologic hypertrophy and that cardiomyocyte secretion of gelsolin may accelerate this negative remodeling response.

Tracking microRNA Processing Signals by Degradome Sequencing Data Analysis.

Degradome sequencing (degradome-seq) was widely used for cleavage site mapping on the microRNA (miRNA) targets. Here, the application value of degradome-seq data in tracking the miRNA processing intermediates was reported. By adopting the parameter “signal/noise” ratio, prominent degradome signals on the miRNA precursors were extracted. For the 15 species analyzed, the processing of many miRNA precursors were supported by the degradome-seq data. We found that the supporting ratio of the “high-confidence” miRNAs annotated in miRBase was much higher than that of the “low-confidence.” For a specific species, the percentage of the miRNAs with degradome-supported processing signals was elevated by the increment of degradome sampling diversity. More interestingly, the tissue- or cell line-specific processing patterns of the miRNA precursors partially contributed to the accumulation patterns of the mature miRNAs. In this study, we also provided examples to show the value of the degradome-seq data in miRNA annotation. Based on the distribution of the processing signals, a renewed model was proposed that the stems of the miRNA precursors were diced through a “single-stranded cropping” mode, and “loop-to-base” processing was much more prevalent than previously thought. Together, our results revealed the remarkable capacity of degradome-seq in tracking miRNA processing signals.

MicroR408 regulates defense response upon wounding in sweet potato.

MiRNAs play diverse roles in plant development and defense responses by binding to their mRNA targets based on sequence complementarity. Here, we investigated a wound-related miR408 and its target genes in sweet potato (Ipomoea batatas) by small RNA deep sequencing and transcriptome analysis. The expression patterns of miR408 and the miR408 precursor were significantly repressed by wounding and jasmonate (JA). In contrast, expression of the putative target genes IbKCS (3-ketoacyl-CoA synthase 4), IbPCL (plantacyanin), and IbGAUT (galacturonosyltransferase 7-like) of miR408 was increased following wounding, whereas only IbKCS was increased after JA treatment. Target cleavage site mapping and Agrobacterium-mediated transient assay demonstrated that IbKCS, IbPCL, and IbGAUT were the targets of miR408. The expression of miR408 target genes was repressed in transgenic sweet potatoes overexpressing miR408. These data indicated a relationship between miR408 and its target genes. Notably, miR408-overexpressing plants showed a semi-dwarf phenotype and attenuated resistance to insect feeding, while transgenic plants overexpressing IbKCS exhibited more insect resistance than plants overexpressing only the empty vector. Collectively, sweet potato reduces the abundance of miR408 upon wounding to elevate the expression of IbKCS, IbPCL, and IbGAUT. The expression of IbKCS enhances the defense system against herbivore wounding.

Proteomic profiling of the proteolytic events in the secretome of the transformed phenotype of melanocyte-derived cells using Terminal Amine Isotopic Labeling of Substrates

The comprehensive profiling of the repertoire of secreted proteins from cancer cells samples provides information on the signaling events in oncogenesis as well as on the cross-talk between normal and tumoral cells. Moreover, the analysis of post-translational modifications in secreted proteins may unravel biological circuits regulated by irreversible modifications such as proteolytic processing. In this context, we used Terminal Amine Isotopic Labeling of Substrates (TAILS) to perform a system-wide investigation on the N-terminome of the secretomes derived from a paired set of mouse cell lines: Melan-a (a normal melanocyte) and Tm1 (its transformed phenotype). Evaluation of the amino acid identities at the scissile bond in internal peptides revealed significant differences, suggesting distinct proteolytic processes acting in the normal and tumoral secretomes. The mapping and annotation of cleavage sites in the tumoral secretome suggested functional roles of active proteases in central biological processes related to oncogenesis, such as the processing of growth factors, cleavage of extracellular matrix proteins and the shedding of ectopic domains from the cell surface, some of which may represent novel processed forms of the corresponding proteins. In the context of the tumor microenvironment, these results suggest important biological roles of proteolytic processing in murine melanoma secreted proteins.

FCLIP-seq for transcriptomic footprinting of dsRNA-binding proteins: Lessons from DROSHA.

CLIP-seq (crosslinking immunoprecipitation and sequencing) is widely used to map the binding sites of a protein of interest on the transcriptome, and generally employs UV to induce the covalent bonds between protein and RNA, which allows stringent washing. However, dsRNA is inefficiently crosslinked by UV, making it difficult to study the interactions between dsRNA binding proteins and their substrates. A dsRNA endoribonuclease DROSHA initiates the maturation of microRNA (miRNA) by cleaving primary miRNA (pri-miRNA). Despite the importance of DROSHA in miRNA maturation and sequence determination, accurate mapping of DROSHA cleavage sites has not been feasible due to rapid processing, modification, and degradation of the cleaved products in cells. Here, we present a high-throughput sequencing method that allows the mapping of in vivo DROSHA cleavage sites at single nucleotide resolution, termed formaldehyde crosslinking, immunoprecipitation, and sequencing (fCLIP-seq). The fCLIP-seq protocol has been improved significantly over the standard CLIP-seq methods by (1) using formaldehyde for efficient and reversible crosslinking, (2) employing polyethylene glycol and adaptors with randomized sequences to enhance ligation efficiency and minimize bias, and (3) performing ligation after elution, which exposes the RNA termini for efficient ligation. fCLIP-seq successfully captures the nascent products of DROSHA, which allows precise mapping of the DROSHA processing sites. Moreover, from the analysis of the distinctive cleavage pattern, we discover previously unknown substrates of DROSHA. fCLIP-seq is a useful tool to obtain transcriptome-wide information on DROSHA activity and can be applied further to investigate other dsRNA-protein interactions.

Genome-wide Mapping of DROSHA Cleavage Sites on Primary MicroRNAs and Noncanonical Substrates

MicroRNA (miRNA) maturation is initiated by DROSHA, a double-stranded RNA (dsRNA)-specific RNase III enzyme. By cleaving primary miRNAs (pri-miRNAs) at specific positions, DROSHA serves as a main determinant of miRNA sequences and a highly selective gatekeeper for the canonical miRNA pathway. However, the sites of DROSHA-mediated processing have not been annotated, and it remains unclear to what extent DROSHA functions outside the miRNA pathway. Here, we establish a protocol termed "formaldehyde crosslinking, immunoprecipitation, and sequencing (fCLIP-seq)," which allows identification of DROSHA cleavage sites at single-nucleotide resolution. fCLIP identifies numerous processing sites, suggesting widespread end modifications during miRNA maturation. fCLIP also finds many pri-miRNAs that undergo alternative processing, yielding multiple miRNA isoforms. Moreover, we discovered dozens of DROSHA substrates on non-miRNA loci, which may serve as cis-elements for DROSHA-mediated gene regulation. We anticipate that fCLIP-seq could be a general tool for investigating interactions between dsRNA-binding proteins and structured RNAs.

Cleavage and cell adhesion properties of human epithelial cell adhesion molecule (HEPCAM).

Human epithelial cell adhesion molecule (HEPCAM) is a tumor-associated antigen frequently expressed in carcinomas, which promotes proliferation after regulated intramembrane proteolysis. Here, we describe extracellular shedding of HEPCAM at two α-sites through a disintegrin and metalloprotease (ADAM) and at one β-site through BACE1. Transmembrane cleavage by γ-secretase occurs at three γ-sites to generate extracellular Aβ-like fragments and at two ϵ-sites to release human EPCAM intracellular domain HEPICD, which is efficiently degraded by the proteasome. Mapping of cleavage sites onto three-dimensional structures of HEPEX cis-dimer predicted conditional availability of α- and β-sites. Endocytosis of HEPCAM warrants acidification in cytoplasmic vesicles to dissociate protein cis-dimers required for cleavage by BACE1 at low pH values. Intramembrane cleavage sites are accessible and not part of the structurally important transmembrane helix dimer crossing region. Surprisingly, neither chemical inhibition of cleavage nor cellular knock-out of HEPCAM using CRISPR-Cas9 technology impacted the adhesion of carcinoma cell lines. Hence, a direct function of HEPCAM as an adhesion molecule in carcinoma cells is not supported and appears to be questionable.

Transcriptome-Wide Cleavage Site Mapping on Cellular mRNAs Reveals Features Underlying Sequence-Specific Cleavage by the Viral Ribonuclease SOX.

Many viruses express factors that reduce host gene expression through widespread degradation of cellular mRNA. An example of this class of proteins is the mRNA-targeting endoribonuclease SOX from the gamma-herpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV). Previous studies indicated that cleavage of messenger RNAs (mRNA) by SOX occurs at specific locations defined by the sequence of the target RNA, which is at odds with the down-regulation of a large portion of cellular transcripts. In this study, we address this paradox by using high-throughput sequencing of cleavage intermediates combined with a custom bioinformatics-based analysis pipeline to identify SOX cleavage sites across the mRNA transcriptome. These data, coupled with targeted mutagenesis, reveal that while cleavage sites are specific and reproducible, they are defined by a degenerate sequence motif containing a small number of conserved residues rather than a strong consensus sequence. This degenerate element is well represented in both human and KSHV mRNA, and its presence correlates with RNA destabilization by SOX. This represents a new endonuclease targeting strategy, in which use of a degenerate targeting element enables RNA cleavage at specific locations without restricting the range of targets. Furthermore, it shows that strong target selectivity can be achieved without a high degree of sequence specificity.

Cleavage and Cell Adhesion Properties of Human Epithelial Cell Adhesion Molecule (HEPCAM)

Human epithelial cell adhesion molecule (HEPCAM) is a tumor-associated antigen frequently expressed in carcinomas, which promotes proliferation after regulated intramembrane proteolysis. Here, we describe extracellular shedding of HEPCAM at two α-sites through a disintegrin and metalloprotease (ADAM) and at one β-site through BACE1. Transmembrane cleavage by γ-secretase occurs at three γ-sites to generate extracellular Aβ-like fragments and at two ϵ-sites to release human EPCAM intracellular domain HEPICD, which is efficiently degraded by the proteasome. Mapping of cleavage sites onto three-dimensional structures of HEPEX cis-dimer predicted conditional availability of α- and β-sites. Endocytosis of HEPCAM warrants acidification in cytoplasmic vesicles to dissociate protein cis-dimers required for cleavage by BACE1 at low pH values. Intramembrane cleavage sites are accessible and not part of the structurally important transmembrane helix dimer crossing region. Surprisingly, neither chemical inhibition of cleavage nor cellular knock-out of HEPCAM using CRISPR-Cas9 technology impacted the adhesion of carcinoma cell lines. Hence, a direct function of HEPCAM as an adhesion molecule in carcinoma cells is not supported and appears to be questionable.

Substrate Cleavage Profiling Suggests a Distinct Function of Bacteroides fragilis Metalloproteinases (Fragilysin and Metalloproteinase II) at the Microbiome-Inflammation-Cancer Interface

Enterotoxigenic anaerobic Bacteroides fragilis is a significant source of inflammatory diarrheal disease and a risk factor for colorectal cancer. Two distinct metalloproteinase types (the homologous 1, 2, and 3 isoforms of fragilysin (FRA1, FRA2, and FRA3, respectively) and metalloproteinase II (MPII)) are encoded by the B. fragilis pathogenicity island. FRA was demonstrated to be important to pathogenesis, whereas MPII, also a potential virulence protein, remained completely uncharacterized. Here, we, for the first time, extensively characterized MPII in comparison with FRA3, a representative of the FRA isoforms. We employed a series of multiplexed peptide cleavage assays to determine substrate specificity and proteolytic characteristics of MPII and FRA. These results enabled implementation of an efficient assay of MPII activity using a fluorescence-quenched peptide and contributed to structural evidence for the distinct substrate cleavage preferences of MPII and FRA. Our data imply that MPII specificity mimics the dibasic Arg↓Arg cleavage motif of furin-like proprotein convertases, whereas the cleavage motif of FRA (Pro-X-X-Leu-(Arg/Ala/Leu)↓) resembles that of human matrix metalloproteinases. To the best of our knowledge, MPII is the first zinc metalloproteinase with the dibasic cleavage preferences, suggesting a high level of versatility of metalloproteinase proteolysis. Based on these data, we now suggest that the combined (rather than individual) activity of MPII and FRA is required for the overall B. fragilis virulence in vivo.

Interaction of small RNA–8105 and the intron of IbMYB1 RNA regulates IbMYB1 family genes through secondary siRNAs and DNA methylation after wounding

Small RNAs (sRNAs) play important roles in plants under stress conditions. However, limited research has been performed on the sRNAs involved in plant wound responses. In the present study, a novel wounding-induced sRNA, sRNA8105, was identified in sweet potato (Ipomoea batatas cv. Tainung 57) using microarray analysis. It was found that expression of sRNA8105 increased after mechanical wounding. Furthermore, Dicer-like 1 (DCL1) is required for the sRNA8105 precursor (pre-sRNA8105) to generate 22 and 24 nt mature sRNA8105. sRNA8105 targeted the first intron of IbMYB1 (MYB domain protein 1) before RNA splicing, and mediated RNA cleavage and DNA methylation of IbMYB1. The interaction between sRNA8105 and IbMYB1 was confirmed by cleavage site mapping, agro-infiltration analyses, and use of a transgenic sweet potato over-expressing pre-sRNA8105 gene. Induction of IbMYB1-siRNA was observed in the wild-type upon wounding and in transgenic sweet potato over-expressing pre-sRNA8105 gene without wounding, resulting in decreased expression of the whole IbMYB1 gene family, i.e. IbMYB1 and the IbMYB2 genes, and thus directing metabolic flux toward biosynthesis of lignin in the phenylpropanoid pathway. In conclusion, sRNA8105 induced by wounding binds to the first intron of IbMYB1 RNA to methylate IbMYB1, cleave IbMYB1 RNA, and trigger production of secondary siRNAs, further repressing the expression of the IbMYB1 family genes and regulating the phenylpropanoid pathway.

Mapping small DNA ligand hydroxyl radical footprinting and affinity cleavage products for capillary electrophoresis

The mapping of DNA footprints and affinity cleavage sites for small DNA ligands is affected by the choice of sequencing chemistry and end label, and the potential for indexing errors can be significant when mapping small ligand–DNA interactions. Described here is a mechanism for avoiding such errors based on a summary of standard labeling, cleavage, and indexing chemistries and a comparison among them for analysis of these interactions by capillary electrophoresis. The length dependence of the difference between Sanger and Maxam–Gilbert indexing is examined for a number of duplexes of mixed sequence.

Mapping of matrix metalloproteinase cleavage sites on syndecan‐1 and syndecan‐4 ectodomains

Syndecans are transmembrane heparan sulfate proteoglycans with roles in cell proliferation, differentiation, adhesion, and migration. They have been associated with multiple functions in tumour progression, through their ability to interact with a wide range of ligands as well as other receptors, which makes them key effectors in the pericellular microenvironment. Extracellular shedding of syndecans by tumour-associated matrix metalloproteinases (MMPs) may have an important role in tumour progression. Such ectodomain shedding generates soluble ectodomains that may function as paracrine or autocrine effectors, or as competitive inhibitors of the intact proteoglycan. Tumour-associated MMPs are shown here to cleave the ectodomains of human syndecan-1 and syndecan-4. Two membrane proximal regions of both syndecan-1 and syndecan-4 are favoured MMP cleavage sites, six and 15 residues from the transmembrane domain. Other sites are 35-40 residues C-terminal from the heparan sulfate chain substitution sites in both syndecans. The MT1-MMP cleavage sites in syndecan-1 and syndecan-4 were confirmed by site-directed mutagenesis. These findings provide insights into the characteristics of syndecan shedding.