Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

hnRNP D (AUF1)

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_150

Synonyms

Historical Background

AUF1, or hnRNP D (heterogeneous nuclear ribonucleoprotein D), was one of the first trans-acting factors identified that binds AU-rich elements (AREs) within the 3′ UTR (UTR – untranslated region) of many labile mRNAs (Brewer 1991). The importance of AREs in promoting rapid RNA turnover has been well characterized (Guhaniyogi and Brewer 2001). A mechanism was still under investigation when AUF1 was discovered. AUF1 as an ARE-binding protein (AUBP) was first identified in specific fractions of cytosol separated by sucrose gradient fractionation (Brewer 1991). Electrophoretic mobility shift assays (EMSAs) of these fractions revealed 37 and 40 kDa (kD) polypeptides that specifically bound ARE-containing c-  myc mRNA. Cell-free decay assays confirmed c-myc mRNA degradation occurred within the same fractions that included the ARE-binding activity. The aforementioned polypeptides, now referred to as p37 and p40, were reproducibly purified from the post-ribosomal, 130,000 × g supernatant, S130, by poly-U agarose chromatography. These polypeptides could cross-link in specific fashion to radiolabeled ARE-containing RNA substrates, including c-fos and GM-CSF 3′UTRs, with the same efficiency seen in the EMSAs (Brewer 1991). Cross-linking could be abolished by mutating the ARE sequence.

An antibody was generated from the poly-U agarose eluate and affinity-purified (Zhang et al. 1993). It detected p37 and p40, as well as a 45-kD polypeptide. Determination of their subcellular localization by cell fractionation and Western blot showed that p37 and p40 were present in both the nucleus and cytoplasm, while p45 was present exclusively in nuclear fractions, thereby explaining why it wasn’t detected initially in the cytosolic S130 fractions.

Screening of a HeLa cDNA expression library with the affinity-purified p40 antibody allowed molecular cloning of p37, which was named AUF1 (ARE/poly (U)-binding/degradation factor 1) and later designated hnRNP D (Zhang et al. 1993; Wagner et al. 1998). Sequence analysis indicated that p37 was an RNA-binding protein distinct from hnRNP A, B, or C. Additional cDNA cloning and sequencing of genomic clones revealed that alternative splicing of the AUF1 pre-mRNA accounted for p37, p40, and p45, as well as a fourth isoform, p42 (Wagner et al. 1998).

Binding of purified AUF1 to AREs in vitro correlated with rapid decay of the transcript (DeMaria and Brewer 1996). Many short-lived cytokine mRNAs contain one or more AREs in their 3'UTR, generally consisting of repeats of overlapping AUUUA pentamers embedded within U-rich sequence (Guhaniyogi and Brewer 2001). EMSAs with cytoplasmic lysates of monocytes revealed that AUF1 bound to the ARE-containing GROa and IL-1ß chemokine/cytokine mRNAs as part of a multi-subunit complex (Sirenko et al. 1997). In human monocytes, these mRNAs remain very labile until the receipt of various extracellular stimuli, whereupon the bound AUF1 and associated protein complex changes in composition, followed by transcript stabilization.

The above studies, as well as others not included in this discussion for purposes of brevity, indicate an important role for AUF1 in regulating immune responses, as many ARE-containing cytokine mRNAs undergo rapid decay until extracellular signaling pathways indicate a need for their transient stabilization. During this window of short-term stability, cytokines are produced in much greater quantities until they are down-regulated again by rapid mRNA decay. Knockdown of AUF1 in mice adversely affects survival, as the mice are unable to suppress overproduction of proinflammatory cytokines (Lu et al. 2006a). The levels of cytokine transcripts, including TNFα and IL-1ß, remain constitutively high; as such, the mice die of severe endotoxic shock due to the runaway effects of cytokine production.

Characterization of AUF1 Protein Structure and Function

AUF1 Genomic Organization

The hnRNP D/AUF1 gene maps to human chromosome locus 4q21 and encodes four different isoforms by differential splicing of the pre-mRNA; a pseudogene lies on the X chromosome (Wagner et al. 1998; Dempsey et al. 1998). The isoforms differ in their molecular weights due to the inclusion or exclusion of specific exons in their respective mRNAs. All isoforms include a pair of RNA recognition motifs (RRMs) and a glutamine-rich region required for RNA binding and protein–protein interactions, respectively. A peptide sequence required for AUF1 dimerization in the absence of RNA is encoded by exon 1, present in all isoforms. The p40 and p45 isoforms contain an additional 19-amino acid insert encoded by exon 2 that in p40 can be phosphorylated in vitro by glycogen synthase kinase-3ß and protein kinase A (Wilson et al. 2003a). The p42 and p45 isoforms have an additional 49-amino acid insert encoded by exon 7 toward the C-terminal end of the proteins, which in p45, confers protection from ubiquitination and subsequent degradation (Laroia and Schneider 2002).

Properties of AUF1 Isoforms

Differential splicing of exons 2 and 7 confers selective subcellular localization and ARE-binding affinities on the AUF1 isoforms (Zhang et al. 1993; Wagner et al. 1998). P37 and p40 are primarily found in the cytoplasm, while p42 and p45 are primarily nuclear. In terms of RNA-binding, the p37 isoform has the highest affinity for AREs, while p40 has the lowest. P42 has an affinity only slightly lower than p37, while the ARE-affinity of p45, though much lower than p37, is still much higher than p40. Altogether, the four isoforms exhibit an approximate 35-fold range of ARE-binding affinities (Wagner et al. 1998). The two isoforms with the highest binding affinity do not contain the exon 2-encoded amino acids.

The N-terminal exon 2 insert contains a pair of serines that can be reversibly phosphorylated on p40 – Ser 83 and Ser 87 (Wilson et al. 2003a, b). In vivo, the phosphates on the two serines are lost upon activation of various cell signaling pathways, correlating with an increase in ARE-mRNA stability, paralleled by changes in mRNA-protein structure (Fig. 1). In vitro studies indicate that p40 can bind to an ARE substrate as both a dimer and a tetramer (Wilson et al. 2003a). When p40 is phosphorylated, the bound RNA is held in an extended conformation versus a more compact configuration generated by the binding of unphosphorylated p40. In vivo, the extended conformation may allow easier access to the ARE by components of the cellular degradation machinery, subsequently accelerating mRNA decay.
hnRNP D (AUF1), Fig. 1

Phosphorylation of p40 induces changes in AUF1-mRNA conformation and accelerates ARE-mRNA decay. For simplicity, p40 is depicted as a dimer in the absence of other RNA-binding proteins. (a) When Ser 83 and Ser 87 are phosphorylated, the mRNA is held in an open conformation relative to the (b) more compact configuration generated by unphosphorylated bound AUF1. Note: AUF1 can also bind as tetramers to AREs in vitro. Phosphorylated p40 and an open mRNP conformation correlate with increased ARE-mRNA decay

AUF1 Can Compete with PABP in Binding to Polyadenylated Sequences

AUF1 cannot only promote mRNA decay via ARE binding, but may also function in dissociating PABP (poly-A binding protein) from transcript tails, depriving the mRNA of its 3-end protection, and allowing digestion by deadenylases and exosomal proteins. Like PABP, AUF1 can also bind polyadenylated sequences. Affinity chromatography of Hela cytoplasmic lysates using poly(A) resin showed that the AUF1 isoforms could specifically bind the poly(A) sequence and not the control poly(C) resin (Sagliocco et al. 2006). This binding was not due to mRNA tethering, nor the amount of PABP present. Further analysis indicated endogenous AUF1 bound the poly(A) resin independently and non-consecutively of PABP, since PABP knockdown had no effect on the amount of AUF1 bound to the resin (Sagliocco et al. 2006).

In vitro, all recombinant AUF1 isoforms can associate cooperatively and sequentially in oligomeric fashion on an RNA substrate containing a poly(A) sequence 100 nucleotides long (Sagliocco et al. 2006). The presence or absence of an ARE in the substrate sequence does not affect the poly(A) binding affinity of AUF1. However, the presence of AUF1 can displace PABP on the poly(A) substrate via competitive binding in these EMSAs. Increasing AUF1 concentration reduced PABP-poly(A) complexes on the RNA substrate in a dose-dependent manner. Simultaneously, AUF1-poly(A) complexes increased. The above data indicating AUF1-PABP competition for the poly(A) tail is an additional step in regulating mRNA decay/stability.

ARE-mRNA Decay Mediated by AUF1 Is Linked to Translation Initiation Factors

AUF1 not only binds proteins at the 3′ end of the transcript like PABP, but also components of the translation initiation complex located at the 5′ end, hinting that translation also plays a role in ARE-mediated decay. Specifically, eIF4G can bind all AUF1 isoforms strongly in vitro, in the presence or absence of an ARE (Lu et al. 2006b), with p37 having the strongest affinity. Further experiments performed with truncation mutants of p37 (His-tagged) and a GST-tagged fragment of eIF4G in GST pulldown assays identified a 45-amino acid domain within the C-terminus of AUF1 necessary for eIF4G binding.

Besides binding to poly(A) sequences (see above), AUF1 can also directly bind to PABP itself in vitro (Lu et al. 2006b). The interaction can be abrogated by adding an RNA consisting of the TNFα ARE to the reaction, and to a lesser extent, by adding Hsp70, whose induction in vivo can stabilize mRNAs. In fact, AUF1 cannot interact with the ARE and PABP simultaneously in an in vitro assay. Interestingly, p37 and PABP can bind concurrently to eIF4G in the absence of an ARE. If an ARE is present, p37 detaches from PABP, leaving behind a stronger binding interaction between PABP and eIF4G. The dynamic AUF1-eIF4G-PABP complex implicates AUF1 in linking rapid mRNA decay to translation.

AUF1 Can Promote Translation of ARE-Containing mRNAs

Posttranscriptional regulation of gene expression by AUF1 is not limited to accelerating mRNA decay. It can alternatively promote translation of ARE-mRNAs. For example, knocking down AUF1 expression does not affect MYC mRNA levels but rather reduces  MYC protein levels at least threefold (Liao et al. 2007). By contrast, overexpression of the individual AUF1 isoforms, or all in combination, increases MYC protein levels by threefold or greater. As well, the presence of MYC mRNA on polyribosomes increases significantly upon overexpression of AUF1. Further analysis showed that the increase in MYC translation is dependent on the ARE. Together, these observations indicated roles for AUF1 in translational control.

The mechanism by which AUF1 promotes translation likely involves competitive binding with translational repressor/s. For example, TIAR knockdown increases MYC protein levels as well as the amount of AUF1 binding to the mRNA. Likewise, AUF1 knockdown increases the amount of TIAR protein bound to the transcript, resulting in reduced MYC translation. This reciprocal competition is ARE-dependent. However, the increase in MYC protein levels by TIAR knockdown is reversed by knocking down AUF1 simultaneously, with no effect on mRNA levels, suggesting that AUF1 can promote MYC translation directly and not solely by blocking TIAR binding.

AUF1 Binds to AREs as Part of a Multi-subunit Complex

AUF1 binds to ARE-containing mRNAs as part of a multi-subunit complex. As heat shock stabilizes ARE-mRNAs, it should not be surprising that AUF1 forms complexes with heat shock proteins Hsc70, Hsp70, and Hsp27 on polysomal mRNA (Laroia et al. 1999; Sinsimer et al. 2008). Hsp70 binding to AUF1 is concomitant with accumulation of stabilized ARE-mRNAs. Also, since heat shock downregulates the ubiquitin-proteosome pathway, which is necessary for AUF1-mediated mRNA decay, it is likely that ubiquitination and proteolysis of AUF1 itself is required for rapid mRNA degradation.

Other subunits of the ARE-bound protein complex include eIF4G and PABP (poly-A binding protein), which function in translation (Laroia et al. 1999). More recently, lactate dehydrogenase was identified as a protein that directly interacts with AUF1, though the significance is unclear (Pioli et al. 2002). The translation factor eIF4G is largely dissociated from the complex until heat-shock reduces its dissociation; this is accompanied by ARE-mRNA stabilization. The cross talk among subunits of the AUF1-containing complex of proteins and between proteins of various signaling pathways are still being elucidated, but it is apparent that ARE-mRNA decay is regulated by a complex process that is often AUF1-dependent and is affected by the rearrangements and posttranslational modifications of the ARE-bound protein complex.

Summary

AUF1 is a trans-acting factor that binds AU-rich elements within the 3′ UTRs of labile mRNAs. There are four isoforms generated by alternative pre-mRNA splicing: p37, p40, p42, and p45. They differ in their subcellular localizations and RNA-binding affinities, suggesting that each isoform has its own subset of target transcripts. AUF1 is needed to promote decay of many ARE-containing transcripts, not just encoding cytokines, but those encoding oncoproteins and anti-apoptotic factors as well (Lapucci et al. 2002). It does so as part of a multi-subunit, ARE-bound complex (Fig. 2). Its binding partners include Hsp70, Hsc70, Hsp27, eIF4G, PABP, lactate dehydrogenase, and other unidentified proteins. The AUF1-containing complex can also compete with RNA-binding proteins such as TIAR to control ARE-mRNA translation (Liao et al. 2007). AUF1-mediated mRNA decay is a complex process that is affected by heat shock, the ubiquitin-proteosome pathway, and posttranslational modification and/or subunit rearrangement of AUF1 and its associated proteins.
hnRNP D (AUF1), Fig. 2

Model for the regulation of ARE-mRNA decay by AUF1 and several known associating proteins. Hsp70/Hsc70, lactate dehydrogenase (LDH), PABP, and eIF4G are complexed with AUF1 to regulate ARE-mRNA decay. Some subunits may remain dissociated from the complex until needed for increased translation. eIF4G and PABP may promote a circular mRNP complex necessary for translational activation

References

  1. Brewer G. An A + U-rich element RNA-binding factor regulates c-myc mRNA stability in vitro. Mol Cell Biol. 1991;11:2460–6.PubMedPubMedCentralCrossRefGoogle Scholar
  2. DeMaria C, Brewer G. AUF1 binding affinity to A + U-rich elements correlates with rapid mRNA degradation. J Biol Chem. 1996;271:12179–84.PubMedCrossRefGoogle Scholar
  3. Dempsey L, Li M, DePace A, Bray-Ward P, Maizels N. The human HNRPD locus to 4q21 and encodes a highly conserved protein. Genomics. 1998;49:378–84.PubMedCrossRefGoogle Scholar
  4. Guhaniyogi J, Brewer G. Regulation of mRNA stability in mammalian cells. Gene. 2001;265:11–23.PubMedCrossRefGoogle Scholar
  5. Lapucci A, Donnini M, Papucci L, Witort E, Tempestini A, Bevilacqua A, et al. AUF1 is a bcl-2 A + U-rich element-binding protein involved in bcl-2 mRNA destabilization during apoptosis. J Biol Chem. 2002;277:16139–46.PubMedCrossRefGoogle Scholar
  6. Laroia G, Schneider R. Alternate exon insertion controls selective ubiquitination and degradation of different AUF1 protein isoforms. Nucleic Acids Res. 2002;30:3052–8.PubMedPubMedCentralCrossRefGoogle Scholar
  7. Laroia G, Cuesta R, Brewer G, Schneider R. Control of mRNA decay by heat-shock-ubiquitin-proteosome pathway. Science. 1999;284:499–502.PubMedCrossRefGoogle Scholar
  8. Liao B, Hu Y, Brewer G. Competitive binding of AUF1 and TIAR to MYC mRNA controls its translation. Genes Dev. 2007;20:3174–84.Google Scholar
  9. Lu J, Sadri N, Schneider R. Endotoxic shock in AUF1 knockout mice mediated by failure to degrade proinflammatory cytokine mRNAs. Genes Dev. 2006a;20:3174–84.PubMedPubMedCentralCrossRefGoogle Scholar
  10. Lu J, Bergman N, Sadri N, Schneider R. Assembly of AUF1 with eIF4G-poly(A) binding protein complex suggests a translation function in AU-rich mRNA decay. RNA. 2006b;12:883–93.PubMedPubMedCentralCrossRefGoogle Scholar
  11. Pioli P, Hamilton BJ, Connolly J, Brewer G, Rigby W. Lactate dehydrogenase is an AU-rich element-binding protein that directly interacts with AUF1. J Biol Chem. 2002;277:35738–45.PubMedCrossRefGoogle Scholar
  12. Sagliocco F, Laloo B, Cosson B, Laborde L, Castroviejo M, Rosenbaum J, et al. The ARE-associated factor AUF1 binds poly(A) in vitro in competition with PABP. Biochem J. 2006;400:337–47.PubMedPubMedCentralCrossRefGoogle Scholar
  13. Sinsimer K, Gratacós F, Knapinska A, Lu J, Krause C, Wierzbowski A, et al. Chaperone Hsp27, a novel subunit of AUF1 protein complexes, functions in AU-Rich element-Mediated mRNA decay. MCB. 2008;28:5233–7.CrossRefGoogle Scholar
  14. Sirenko O, Lofquist A, DeMaria C, Morris J, Brewer G, Haskill JS. Adhesion-dependent regulation of an A + U-rich element-binding activity associated with AUF1. Mol Cell Biol. 1997;17:3898–906.PubMedPubMedCentralCrossRefGoogle Scholar
  15. Wagner B, DeMaria C, Sun Y, Wilson G, Brewer G. Structure and genomic organization of the human AUF1 gene: alternative pre-mRNA splicing generates four protein isoforms. Genomics. 1998;48:195–202.PubMedCrossRefGoogle Scholar
  16. Wilson G, Lu J, Sutphen K, Suarez Y, Sinha S, Brewer B, et al. Phosphorylation of p40AUF1 regulates binding to A + U-rich mRNA destabilizing elements and protein-induced changes in ribonucleoprotein structure. J Biol Chem. 2003a;278:33039–48.PubMedCrossRefGoogle Scholar
  17. Wilson G, Lu J, Sutphen K, Sun Y, Huynh Y, Brewer G. Regulation of A + U-rich element-directed mRNA turnover involving reversible phosphorylation of AUF1. J Biol Chem. 2003b;278:33029–38.PubMedCrossRefGoogle Scholar
  18. Zhang W, Wagner B, Ehrenman K, Schaeffer A, DeMaria C, Crater D, et al. Purification, characterization, and cDNA cloning of an AU-rich element RNA-binding protein, AUF1. Mol Cell Biol. 1993;13:7652–65.PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Department of Molecular Genetics, Microbiology and ImmunologyUniversity of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical SchoolPiscatawayUSA