Molecular cloning and characterization of the human RNase κ, an ortholog of Cc RNase

A novel protein family, designated hereafter as RNase κ (kappa) family, has been recently introduced with the characterization of the specific Cc RNase, isolated from the insect Ceratitis capitata. The human ortholog of this family consists of 98 amino acids and shares > 98% identity with its mammalian counterparts. This RNase is encoded by a single-copy gene found to be expressed in a wide spectrum of normal and cancer tissues. The cDNA of the human ribonuclease has been isolated and subcloned into a variety of prokaryotic expression vectors, but most efforts to express it caused a severe toxic effect. On the other hand, the expression of the human RNase by the use of the methylotrophic yeast Pichia pastoris system resulted in the production of a highly active recombinant enzyme. Using a 30-mer 5′-end-labeled RNA probe as substrate, the purified enzyme seems to preferentially cleave ApU and ApG phosphodiester bonds, while it hydrolyzes UpU bonds at a lower rate. Based on amino acid sequence alignment and substrate specificity data, as well as the complete resistance of the recombinant protein to the placental ribonuclease inhibitor, we concluded that the human RNase κ is a novel endoribonuclease distinct from other known ribonucleases

A subtle alternative splicing event gives rise to a widely expressed human RNase k isoform.

Subtle alternative splicing leads to the formation of RNA variants lacking or including a small number of nucleotides. To date, the impact of subtle alternative splicing phenomena on protein biosynthesis has been studied in frame-preserving incidents. On the contrary, mRNA isoforms derived from frame-shifting events were poorly studied and generally characterized as non-coding. This work provides evidence for a frame-shifting subtle alternative splicing event which results in the production of a novel protein isoform. We applied a combined molecular approach for the cloning and expression analysis of a human RNase κ transcript (RNase κ-02) which lacks four consecutive bases compared to the previously isolated RNase κ isoform. RNase κ-02 mRNA is expressed in all human cell lines tested end encodes the synthesis of a 134-amino-acid protein by utilizing an alternative initiation codon. The expression of RNase κ-02 in the cytoplasm of human cells was verified by Western blot and immunofluorescence analysis using a specific polyclonal antibody developed on the basis of the amino-acid sequence difference between the two protein isoforms. The results presented here show that subtle changes during mRNA splicing can lead to the expression of significantly altered protein isoforms

Cc RNase: the Ceratitis capitata ortholog of a novel highly conserved protein family in metazoans

Complementary DNA encoding a protein, designated Cc RNase, was isolated from the insect Ceratitis capitata. Deduced amino acid sequence analysis demonstrates that the Cc RNase has strong sequence homology with other uncharacterized proteins predicted from EST sequences belonging to different animal species, therefore defining a new protein family, which is conserved from Caenorhabditis elegans to humans. Phylogenetic analysis data in addition to extensive homolog searches in all available complete genomes suggested that all family members are true orthologs. Proteins belonging to this family are composed of 95–101 amino acids. The C.capitata orthologous protein was expressed in Escherichia coli. Despite the fact that the amino acid sequence of Cc RNase does not share any significant similarities with other known ribonucleases, our data give strong evidence in support of the assignment of enzymatic activity to the recombinant protein. The expressed molecule exhibits ribonucleolytic activity against poly(C) and poly(U) synthetic substrates, as well as rRNA. It is also demonstrated that expression of Cc RNase in E.coli inhibits growth of the host cells

Schematic representation of the hybrid capture protocol for the selection of RNase κ-02 alternative transcript.

<p>The method consists of the following steps: (i) Reverse transcription of poly(A)⁺ mRNA and construction of a single stranded cDNA library. (ii) Hybridization of cDNA molecules with a biotinylated single stranded RNase κ specific DNA probe immobilized on streptavidin magnetic beads. (iii) Complete digestion of the hybrids with an appropriate restriction enzyme. (iv) PCR amplification of the selected target cDNA.</p

Schematic representation of the human RNase κ gene, its derived mRNA isoforms and amino acid sequence alignment of the protein isoforms.

<p>(A) White boxes represent gene exons and the horizontal bold lines introns. In mRNAs, the black parts of the rectangles demonstrate the protein-coding regions and the white the non-coding regions. The grey line represents the GTTG sequence that is present in RNase κ-01 isoform. The positions of ATG initiation codons are marked on both isoforms. (B) Amino acid sequences are numbered on the right and the identical portion of the two proteins is marked in grey frame.</p

Immunofluorescence microscopy analysis of RNase κ-02.

<p>HEK-293 cells (A) and RNAi depleted RNase κ-02 HEK-293 cells (B) were treated with K02N specific polyclonal antibody and protein – primary antibody complexes were visualized with rabbit matched Alexa-488 secondary antibody. DNA was stained with propidium iodide (B,E). Merged image of the two stains (C,F).</p

RNase κ-01/RNase κ-02 mRNA ratio in human cell lines.

<p>(A) A histogram of RNase κ mRNA isoforms ratio. Total RNA isolated from 12 human cell lines was reverse transcribed and amplified for 10 cycles by regular PCR. Equal amount of the PCR products from each reaction were incubated in the presence or in the absence of FatI and the samples were re-amplified by Real Time PCR. The comparative ΔC_T analysis performed as described in Materials and Methods resulted in the relative quantification of RNase κ mRNA isoforms. Error bars denote the standard error of the mean of triplicate reactions performed three times for each cell line. (B) The Real Time PCR products from the digested samples were overnight incubated with FatI and analyzed by electrophoresis in a 2% agarose gel. In a parallel experiment, 200 ng of RNAse κ-01 cDNA amplified by the same primers were digested under the same conditions as a control reaction.</p