Research article
Computational analysis and in vivo validation of a microRNA encoded by the IBTK gene, a regulator of B-lymphocytes differentiation and survival

https://doi.org/10.1016/j.compbiolchem.2009.08.001Get rights and content

Abstract

MicroRNAs (miRNAs) are small single-stranded RNA molecules that play an essential role in the regulation of gene expression and cell physiology. Gene rearrangements occurring in the miRNA sequence are associated with cancer. The IBTK genetic locus is located in the genomic sequence 6q14.1 that undergoes chromosomal aberration in lymphoproliferative disorders. The IBTK gene encodes the proteins IBtk-α, β and γ that regulate the B cell receptor signalling through Bruton's tyrosine kinase, which promotes B cell survival and differentiation. Pro-MirII-based analysis predicted four precursors of microRNAs (pre-miR) encoded by introns 17, 21, 26 and the 3′ un-translated region of the IBTK gene. Pre-miR-IBTK3, which was encoded by intron 26, was the effective substrate of RNase III Dicer in vitro as well as the precursor of an IBtk miRNA generated in vivo. By CLUSTALW-based analysis, pre-miR-IBTK3 homologues were found in Pan troglodytes, Pongo pygmaeus and Macaca mulatta, suggesting an evolutionary conserved function in primates.

Introduction

MicroRNAs (miRNAs) are single-stranded RNA molecules of 21–23 nucleotides in length that act through base pairing to partially complementary sequences of the 3′ un-translated region (3′UTR) of targeted mRNAs. This event destabilizes the target transcript or inhibits the translation of the related proteins (Ambros, 2001). Similarly to transcription factors, miRNAs play an essential role in the regulation of gene expression (Bartel, 2009).

MicroRNAs result from the processing of the primary transcript (pri-miR) generated by RNA polymerase II (Lee et al., 2004) or RNA polymerase III (Pfeffer et al., 2005). Once capped and poly-adenylated (Cai et al., 2004), the pri-miR enters a microprocessor complex (500–650 kDa) consisting of Drosha, an RNase III endonuclease, and an essential cofactor DGCR8/Pasha protein containing two double-stranded RNA binding domains (Denli et al., 2004, Gregory et al., 2004). The pri-miR is cleaved by Drosha to generate the precursor of miRNA (pre-miR) consisting of a 60–100 nucleotides stem–loop sequence with a 5′ phosphate and two nucleotides 3′ overhangs. The pre-miR is transported to the cytoplasm by Exportin-5, a member of the Ran transport receptor family (Yi et al., 2003). Finally, the cytosolic RNase III Dicer cleaves the pre-miR stem–loop structure to generate the mature single-stranded miRNA (Grishok et al., 2001). The miRNA is incorporated in the cytosolic effector complex, named RNA-Induced Silencing Complex (RISC), where it anneals to the 3′UTR of targeted transcripts by complementary base pairs causing the destabilization or translational inhibition of the target mRNA (Gregory et al., 2005).

MicroRNAs play an essential role in the regulation of cell survival and differentiation (Schickel et al., 2008). Indeed, gene rearrangements causing miRNA mutations result in deregulated gene expression that has been frequently associated with human cancers (Calin and Croce, 2006, Calin and Croce, 2007a, Calin and Croce, 2007b, Li et al., 2008). This evidence suggests that tumor suppressors and oncogenes may harbour miRNAs that target specific mRNAs and thus deregulate cell proliferation and survival.

We have recently characterized the Inhibitor of Bruton's tyrosine kinase (IBtk) gene (accession number AL050333) that encodes three adaptor proteins in cell signalling (Spatuzza et al., 2008) The IBTK gene is 77.58 kb and includes 29 exons with two promoters and transcriptional start sites that result in the expression of three IBTK transcripts, named IBTKα, IBTKβ and IBTKγ (Spatuzza et al., 2008). IBtkγ has been characterized as a ligand inhibitor of Tec kinases, such as Btk, Itk, and Akt, which regulate signal transduction upon specific stimuli (Spatuzza et al., 2008, Liu et al., 2001). As inhibitor of proliferation, IBTK is a candidate tumor suppressor gene. Consistently, the human IBTK gene is located in the genomic sequence 6q14.1, which is a hot spot of chromosomal rearrangements in lymphoproliferative disorders (Mitelman et al., 1997).

In this study, we have addressed whether the IBTK gene might play a role in transcription regulation as source of miRNAs. By bioinformatics analysis, we identified four putative precursors of miRNA (pre-miR) encoded by three distinct introns and the 3′ un-translated region (3′UTR) of the IBTK gene. Of them, only the pre-miR-IBTK3 encoded by intron 26 occurred in vivo, and was the effective substrate of RNase III Dicer. The presence of homologous in other primates suggests an evolutionary conserved role of pre-miR-IBTK3. Based on this evidence, IBTK miRNA is a novel member of the wide miRNAs genomic network.

Section snippets

Prediction of potential miRNAs

Search for putative miRNAs generated by the genetic locus IBTK was performed by Pro-MirII software (http://cbit.snu.ac.kr/∼ProMiR2/index.php). The pre-miR-IBTK nucleotide sequences were predicted according to the following cut-off values: window size: 100 base pairs; shift size: 10 nucleotides; Pro-Mir values: 0.017; conservation score  0; free energy (ΔG)  −20; G–C ratio = 0.3–0.7; entropy  1.8.

DNA plasmids

To generate T7 expression plasmids of pre-miR-IBTK1, pre-miR-IBTK2, pre-miR-IBTK3 and pre-miR-IBTK4, the

Results

To identify putative pre-miRs encoded by the IBTK gene, we performed an in silico analysis of the genomic nucleotide sequence of the IBTK genetic locus by using the Pro-MirII software that allows the probabilistic prediction of clustered, un-clustered, conserved and un-conserved (Nam et al., 2006). We identified four potential miRNAs located in the introns 17, 21, 26 and in the 3′UTR of the IBTK gene, named pre-miR-IBTK1, pre-miR-IBTK2, pre-miR-IBTK3, pre-miR-IBTK4, respectively, according to

Discussion

The IBTK genetic locus encodes the IBtk proteins α, β, γ (Spatuzza et al., 2008). We previously characterized IBtkγ as a repressor of Btk (Spatuzza et al., 2008, Liu et al., 2001). Btk is a Tec tyrosine kinase required for B-lymphocytes development and differentiation (Halcomb et al., 2007). Mutations of the BTK gene inactivating the Btk activity caused X-linked agammaglobulinemia in humans (Tsukada et al., 1993) and X-linked immunodeficiency in mice (Vinuesa et al., 2001). In mature

Acknowledgements

We thank Dr. M. Sardiello and Dr. V.A. Gennarino for helpful suggestions. This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC), MIUR-PRIN, MIUR-FIRB and ISS to G.S. and I.Q.

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