Characterization of a novel transcript variant of human STAU 1 gene *

Human STAU1 is one member of the family of double-stranded RNA (dsRNA)-binding proteins. It is thought to function in transporting mRNA, controlling translation and eliciting mRNA decay in neurons, and to function in infection of influenza virus and human immunodeficiency virus type 1 (HIV-1). Four transcripts coding two isoforms have been identified before. In this study, we have isolated a novel transcript of STAU1, coding a novel isoform that has six amino acids more (SFPLKQ) than isoform a. In order to examine the tissue distribution of this novel isoform, we have performed RT-PCR experiments and the analysis showed that it was highly expressed in heart, liver, kidney and pancreas.


INTRodUCTIoN
Drosophila Staufen, homologous human STAU1, is the first RNA-binding protein proven to play a role in RNA localization.Staufen and its homologous genes play a key role in many biological phenomena.One of the best known examples is determination of the anteroposterior axis during the early development of Drosophila melanogaster.Staufen plays a role in the transportation and location of bicoid and oskar mRNAs to the anterior and posterior poles, respectively (Schuldt et al., 1998).The specfic localization of mRNAs is also essential in the definition of cell asymmetries in cell division and differentiation.Staufen transports prospero mRNA to basal area of fly mitotic neuroblasts (Li et al., 1997).Cell comunication events are also affected by the localization of specific mRNAs.Human Stau1 can interact with many proteins and locates in the dendrites of differentiated neuroblasts (Kiebler & DesGroseillers, 2000).Human STAU1 functions in infection by influenza virus and human immunodeficiency virus type 1 (HIV-1).It can interact with NS1 protein of influenza virus in infected cells (Falcón et al., 1999), and it can also interact with the NC domain of HIV-1 pr55 Gap and may be make the virus generate infectious viral particles (Chatel-Chaix et al., 2004).In addition to these biological processes, Drosophila Staufen function in the translation derepression of oskar mRNA once the mRNA has been located (Micklem et al., 2000).Human STAU1 causes the decay of many mRNAs through binding Upf1 and interacting with the 3' untranslated regions of mRNAs (Kim et al., 2005).
Four transcript variants resulting from human STAU1 gene and encoding two isoforms (a, b) with different N-terminal ends have been described (Marión et al., 1999).Three of these variants encode the same isoform (a), however, differ in their 5' UTR.In contrast to Drosophila Staufen, human STAU1 has only four dsRBDs.However, the significance of the various transcripts is not known today.In a recent study, we have isolated a novel transcript variant of STAU1 through a large-scale sequencing analysis of the 18-week human fetal brain cDNA library.We termed it STAU1 transcript variant T5 (isoform c).Here, we report the cloning and initial characterization of this new transcript variant, together with data in its genomic structure and mRNA tissue distribution.

MATeRIALS ANd MeTHodS
The cloning and bioinformatics analysis.cDNA library construction and DNA sequencing were performed as previously described (Li et al., 2005).DNA sequence homology searches and comparisons were performed using BLAST-N and BLAST-X at the National Center for Biotechnology Information (NCBI) network service (http:// www.ncbi.nlm.nih.gov/blast).BLAST-N searching in the human genome was performed to identify the chromosomal localization and the gene structure of this novel gene.The predicted amino-acid sequence of the STAU1 T5 was compared against the profile entries to find the occurrence of known profiles (http://www.expasy.ch/pfscan).To identify the chromosomal localization and the gene structure, multiple alignments were performed by the GeneDoc program (http://www.psc.edu/biomed/genedoc/).Other databases from Genbank, Swiss Pro, PDB, and EXPASY were also used.The software includes Gene Runner, Primer Premier 5.0 et al.
Assessment of human STAU1 T5 mRNA tissue distribution.Human Multiple Tissue cDNA (MTC) panels (CLONTECH) were used as PCR templates according to the manufacturer's protocol.Thirty six PCR cycles for STAU1 T5 and 30 PCR cycles for G3PDH (as control) were performed using Taq Plus polymerase (Sangon) in the following program: 0.5 min at 94 o C, 0.5 min at 65 o C, 1.0 min at 72 o C. The PCR products of STAU1 T5 and G3PDH were then electrophoresed on a 2% agarose gel.PCR primers are indicated from 5' to 3' as the following: STAU1 T5 sense CTCTCTCGGCTCCCGCTTCCTTT; STAU1 T5 antisense ACCTGTTTCAGAGGGAAAGACTCG; G3PDH sense: TGAAGGTCGGAGTCAACGGATTT-GGT; G3PDH antisense CATGTGGGCCATGAGGT CCACCAC.The sense and antisense primers of human STAU1 T5 span 688 bp in the cDNA from 61 to 748 bp.

ReSULTS ANd dISCUSSIoN
Through the large-scale cDNA sequencing, we cloned a novel transcript variant from the constructed human fetal brain cDNA library.This cDNA is composed of an open reading frame from nucleotide 363 to 1868, encoding a 502 amino-acid protein with a molecular mass of 55 kDa (Genbank: AY546099) (Fig. 1).The deduced protein shows high identity with the mouse and Drosophila Staufen2 proteins.Alignment was performed between STAU1 T5, other two isoforms in human, and its orthologs from mouse and Drosophila.Like other Staufens, the deduced protein of T5 also contains four dsRNA-binding domains (dsRBDs) (Fig. 2).Universally, dsRBD2 contains an insertion sequence at an identical position in all species, and the domain does not bind RNA when this insertion is deleted.Surprisingly, we found another insertion of 18 bp in the sequence, which is deduced into six amino acids between dsRBD1 and dsRBD2.This new insertion may suggest a new kind of mechanism or method of RNA localization during the cell cycle or embryo development.Moreover, through ortholog analysis (not shown), the insertion of the 18 bp sequence is also present in certain Staufen protein in Drosophila, which suggests that this protein may be conserved in evolution and that this insertion may have something to do with the specificity of its binding in different tissues of human and Drosophila.However, on its own Staufen binds mRNA without apparent specificity, indicating that the specificity might be provided by auxiliary factors.
Four transcript variants resulting from alternative splicing of the STAU1 gene and encoding two isoforms with different N-terminal ends have been reported.Three of these variants encode the same isoform, however, differ in their 5' UTR.

Figure 1 .
Figure 1.The transcript variant cdNA and deduced amino-acid sequences of STAU1 T5.The nucleotides are numbered at the left.The extra amino-acid residues of Stau1 T5 compared to other isoforms are boxed.

Figure 3 .
Figure 3. Tissue distribution of human STAU1 T5 mRNA.Reverse transcription-PCR analysis of human cDNA for STAU1 T5 and G3PDH (as a control).Prenormalized cD-NAs from sixteen human adult tissues were purchased from CLONTECH and employed as templates in PCR reactions containing STAU1 T5 and G3PDH-specific primers described in Materials and Methods.

Table 1 . The exon-intron analysis of the human STAU1 T5 gene.
ttccttaaagGTGTGGGAGGIntron and exon nucleotide sequences are shown in lower-case and upper-case letters, respectively.Bold italics lettering indicates donor and acceptor splice site.