Identification and characterization of PNRC splicing variants

Abstract

Nuclear receptor (NR) dependent transcriptional action requires recruitment of diverse factors characterized as coregulators. PNRC (proline-rich nuclear receptor coregulatory protein) is a member of coregulators that are capable of potentiating the transcriptional activity of NRs. Here we identified three human PNRC splicing variants designated PNRC1c, PNRC1d and PNRC1f. PNRC1c and PNRC1f are generated through alternative recognition of the 3'-splice site in exon 1, leading to in-frame deletion of 79 amino acids aa) and an altered reading frame, respectively. PNRC1d is generated through the alternate promoter usage and forms a truncated protein containing C-terminus 142 aa of full-length PNRC. These isoforms differ in their abilities to bind NRs and potentiate NR mediated transcriptions. Moreover, PNRC1d can modulate the activity of full-length PNRC in enhancing ER mediated transcription. Our results suggest PNRC exists as functionally distinct isoforms and alternative splicing serves as a regulatory mechanism of PNRC coactivator activity.

PNRC accumulates in the nucleolus by interaction with B23/nucleophosmin via its nucleolar localization sequence

“…As shown in Fig. 1B, GFP was distributed throughout the cell, whereas GFP-tagged PNRC was predominantly localized in the nucleus with concentrated foci, which is consistent with our previous result [13][14]. The foci are likely nucleoli because they have the characteristic condensed structure of nucleoli visible under the light microscope [15].…”

“…PNRC exerts its functions mainly in the nucleus, and our previous studies also show that it localizes in the nucleus [5,13]. There is a putative nuclear localization sequence (NLS) within PNRC, which is located at position 94-101 ( 94 PKKRRKKK 101 ) [10], and recently we have confirmed that it is the real NLS of PNRC [14].…”

“…All recombinant constructs were verified by DNA sequencing. Vectors expressing GFPtagged wild-type PNRC and GFP-tagged PNRC 186-327 were prepared as previously described [13]. To generate the vectors expressing GFP-tagged PNRC 1-185 and GFP-tagged NLS-PNRC 186-327 , the DNA fragments coding PNRC 1-185 and NLS-PNRC 186-327 were amplified by PCR using pEGFP-C1-PNRC as the template and the following oligonucleotides as the primers (PNRC 1-185 : 5'-GATCTCGAGCTATGA CTGTCGTCTCCGTCCCG-3' and 5'-GCCGAATTCTCACTTTGATTTTAAAACC TCTT-3'; NLS-PNRC 186-327 : 5'-GATCTCGAGCTCCGAAGAAGCGGCGAAAGAA GAAGATGGGAAAATCGGA GAA -3' and 5'-GCGTGATCACTAAGTTTGAACTT TGAGGAG-3').…”

BH3-only proteins in apoptosis and beyond: an overview

“…Alternative splicing of the BH3-only members also appear to contribute to the diversity of these proteins and regulate their functions. For example, multiple splice isoforms were described for BIM (Marani et al, 2002; O’Connor et al, 1998; U et al, 2001), BID (Renshaw et al, 2004), NOXA (Wang and Sun, 2008), BNIP1 (Zhang et al, 1999), BMF (Morales et al, 2004). Some of these splice isoforms (like in BIM) retain the BH3-domain, while in other (like in BMF and NOXA) it is spliced out.…”

Regulation of stressed-induced cell death by the Bcl-2 family of apoptotic proteins

“…The HUGO approved name is PMAIP1 (phorbol-12-myristate-13-acetate-induced protein 1). The human Noxa gene is composed of three exons and two introns, which encodes a 54 amino acid protein containing one BH3 domain (Wang & Sun, 2008). The transcript encoding the NOXA protein consists of exon 1 and 3.…”