A chicken hnRNP of the A/B family recognizes the single‐stranded d(CCCTAA)_n telomeric repeated motif

Abstract

With the aim of identifying proteins able to interact with the C-rich single-stranded telomeric repeated motif, three nuclear polypeptides, CBNPa, CBNPb and CBNPg, with apparent mobilities in SDS/PAGE of 38, 44 and 55 kDa, respectively, were isolated from mature chicken erythrocytes by affinity chromatography. In situ UV-cross-linking experiments demonstrated that CBNPa and CBNPg interact directly with the telomeric d(CCCTAA) n repeat, whereas CBNPb does not. Moreover, they provided information on the protein components responsible for each electrophoretic mobility-shift assay signal. Ion spray and matrix-assisted laser desorption ionization MS allowed us to identify CBNPa with single-stranded D-box-binding factor (ssDBF), a protein previously characterized as a transcription factor belonging to the A/B family of heterogeneous nuclear ribonucleoproteins, and CBNPb with an isoform of this protein containing an extra exon. Similarly, CBNPg was shown to be probably the chicken homolog of hnRNP K, a ribonuclear protein able to bind to polyC oligonucleotides. The relation of CBNPa (i.e. ssDBF), CBNPb and CBNPg to a number of similar proteins in the protein and nucleotide sequence databank is discussed. A rather diversified spectrum of functional roles has been assigned to some of these proteins despite the strong sequence homology among them.Keywords: heterogeneous ribonucleoproteins (hnRNPs); nuclear proteins; ssDNA recognition; telomeric C-rich motif.Since the discovery of the special features of repetitive DNA which constitutes the majority of telomeres of eukaryotic organisms, and of its specific replication machinery, considerable attention has also been directed to identifying and characterizing nuclear proteins that specifically bind to this DNA, in the normal Watson±Crick duplex form, as well as to the protruding single-stranded G-rich 3 H overhang at the telomere terminus. Many proteins such as TRF1 and TRF2 that bind to duplex telomeric DNA in mammalian cells [1] and Rap1p in yeast [2] have been reported to be involved in telomere length maintenance and regulation of telomerase activity. In unicellular organisms, several proteins, such as ab protein from Oxytricha , also bind to the single-stranded G-rich telomeric motif, although their function has not been fully ascertained. The last of these, however, is the first ssDNA-binding protein shown to be directly involved in mammalian telomere biogenesis, suggesting a possible mechanism by which telomere length can be modulated [13]. Much less attention has been given to identifying nuclear components able to recognize the complementary single-stranded C-rich DNA repeat. Interestingly, a protein from Trypanosoma, ST-1, binds to the telomeric double-stranded repeat as well as to its singlestranded C-rich component [14]. In vertebrates, the nuclear protein from rat hepatocytes, qTBP42, has been shown to recognize each of the single-stranded forms of the telomeric repeat [9]. Several proteins that interact with polypyrimidine ssDNA have been described. They includ...

Bovine Cytosolic 5′-Nucleotidase Acts through the Formation of an Aspartate 52-Phosphoenzyme Intermediate

“…Raw data were analyzed by using computer software provided by the manufacturer and are reported as average masses. Assignments of the recorded mass values to individual peptides were performed on the basis of their molecular mass as previously described (21).…”

“…Gel bands were reduced, alkylated, and digested in situ with trypsin as previously reported (21). The resulting digests were separated by reverse phase-HPLC as illustrated in Fig.…”

“…Protein bands were excised, and gel pieces were tritured, washed with water, reduced with dithiothreitol and carboxamidomethylated (21). Gel pieces were equilibrated in 0.4% NH 4 HCO 3 and finally digested in situ with trypsin in 0.4% NH 4 HCO 3 , pH 8.5, at 37°C for 18 h, (enzyme/substrate ratio 1:30 w/w).…”

The Folding and Unfolding Kinetics of the i‐Motif Structure Formed by the C‐Rich Strand of Human Telomere DNA

“…[1][2][3][4][5][6] These sequences are widely dispersed in genomic DNA, [7] and proteins have been found that interact selectively with such sequences, possibly in their i-motif form; [8][9][10][11][12] this suggests that these sequences and the relevant i-motif structures might be involved in biological processes. [13] It has been reported, for example, that the i-motif upstream of the insulin gene might facilitate transcription.…”

Characterization of hnRNP A3 isoforms in The A2RE-proteome

“…Subsequent studies showed that hnRNP A1 protect the G-rich overhang region from degradation (Dallaire, Dupuis, Fiset, & Chabot, 2000), while hnRNPs D, K and E prefer the hnRNPrich telomeric sequence (Bandiera et al, 2003;Marsich, Bandiera, Tell, Scaloni, & Manzini, 2001). hnRNP D and its isoforms not only bind to ss oligonucleotides but also disrupt G-G pairing, facilitating telomerase access (Eversole & Maizels, 2000).…”