Physiological networks and disease functions of RNA-binding protein AUF1.

Advanced Review

Physiological networks and disease functions of RNA-binding protein AUF1 Ashleigh E. Moore, Devon M. Chenette, Lauren C. Larkin and Robert J. Schneider∗ Regulated messenger RNA (mRNA) decay is an essential mechanism that governs proper control of gene expression. In fact, many of the most physiologically potent proteins are encoded by short-lived mRNAs, many of which contain AU-rich elements (AREs) in their 3′ -untranslated region (3′ -UTR). AREs target mRNAs for post-transcriptional regulation, generally rapid decay, but also stabilization and translation inhibition. AREs control mRNA turnover and translation activities through association with trans-acting RNA-binding proteins that display high affinity for these AU-rich regulatory elements. AU-rich element RNA-binding protein (AUF1), also known as heterogeneous nuclear ribonucleoprotein D (HNRNPD), is an extensively studied AU-rich binding protein (AUBP). AUF1 has been shown to regulate ARE-mRNA turnover, primarily functioning to promote rapid ARE-mRNA degradation. In certain cellular contexts, AUF1 has also been shown to regulate gene expression at the translational and even the transcriptional level. AUF1 comprises a family of four related protein isoforms derived from a common pre-mRNA by differential exon splicing. AUF1 isoforms have been shown to display multiple and distinct functions that include the ability to target ARE-mRNA stability or decay, and transcriptional activation of certain genes that is controlled by their differential subcellular locations, expression levels, and post-translational modifications. AUF1 has been implicated in controlling a variety of physiological functions through its ability to regulate the expression of numerous mRNAs containing 3′ -UTR AREs, thereby coordinating functionally related pathways. This review highlights the physiological functions of AUF1-mediated regulation of mRNA and gene expression, and the consequences of deficient AUF1 levels in different physiological settings. © 2014 John Wiley & Sons, Ltd. How to cite this article:

WIREs RNA 2014, 5:549–564. doi: 10.1002/wrna.1230

INTRODUCTION

G

ene expression can be controlled at the post-transcriptional level through multiple mechanisms that contribute to the regulation of messenger RNA (mRNA) stability and translation. The impact of regulated mRNA decay is apparent ∗ Correspondence

to: [email protected]

Alexandria Center for Life Sciences, New York University School of Medicine, New York, NY, USA Conflict of interest: The authors have declared no conflicts of interest for this article.

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in genome-wide mRNA analyses, as approximately half of the changes in physiologically inducible gene expression occur at the level of mRNA stability.1 Many cellular factors and mechanisms have been identified in recent years that regulate the rate of mRNA decay in response to physiological stimuli, further highlighting the importance of this level of gene control. One key mechanism by which specific mRNAs are targeted for regulation by post-transcriptional mechanisms is through mRNA cis-acting regulatory elements that typically reside within the 3′ untranslated region (3′ -UTR) of select mRNAs.2

© 2014 John Wiley & Sons, Ltd.

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The AU-rich element (ARE) is one type of cis-acting mRNA element that has been shown to control the stability and translation of many inducible mRNAs, including inflammatory cytokine, growth factor, cell cycle control, and oncogene mRNAs.3 AREs impart their regulatory functions through interaction with trans-acting RNA-binding proteins that display high affinity for AREs. Approximately 20 AU-rich binding proteins (AUBPs) have been identified to date. These RNA-binding proteins have unique and at times opposing effects on the fate of an ARE-mRNA to which they are bound. AUBPs exert their regulatory role by either promoting or impairing rapid mRNA turnover or translation.2 The regulatory capability of these AUBPs is therefore quite far reaching, as it is estimated that approximately 16% of all human protein-coding genes are encoded by mRNAs that contain an ARE sequence within their 3′ -UTR.4 AUF1 has been shown to act as an AREmRNA decay factor, and occasionally as an AREmRNA-stabilizing factor, although the consensus is that it primarily functions to promote rapid mRNA degradation.5 In addition to its post-transcriptional regulatory function, AUF1 has also been proposed to regulate the expression of certain genes and mRNAs at the transcriptional and translational level. AUF1 comprises a family of proteins consisting of four related isoforms derived from a common mRNA precursor by differential splicing of exons 2 and 7, referred to as p37AUF1 , p40AUF1 , p42AUF1 , and p45AUF1 , based on their molecular weights. Although each isoform contains common protein domains critical for RNA binding, studies have identified overlapping and unique functions for the different protein isoforms conferred by the inclusion of alternately spliced exons, as well as widely different expression levels, subcellular localization, and post-translational modifications. AUF1 has the ability to exhibit pleiotropic effects on gene expression owing to its capability to interact with many types of ARE-mRNAs, as well as its capacity to bind DNA, presumably at regulatory sites, and therefore impacts numerous biological pathways at different levels of gene regulation.6,7 AUF1 may serve as a central node of regulation by coordinating both the stability and, in certain circumstances, the transcription of groups of mRNAs in biochemical pathways, thereby integrating complex regulatory circuits to maintain tissue homeostasis. This review examines the contribution of AUF1 proteins in regulating gene expression, through multiple mechanisms of action, on complex physiological 550

networks and its likely involvement in different disease entities that depend on tissue context and level of its expression.

THE AUF1 FAMILY OF PROTEINS AUF1 was first identified using an in vitro mRNA decay system to characterize candidate factors that contribute to the instability of cytokine and proto-oncogene mRNAs. Two polypeptides of 37 and 40 kDa were isolated and characterized as AUF1 proteins, which were found to be present in a cytoplasmic fraction that contributed to rapid c-Myc mRNA degradation.8–10 A related 45-kDa polypeptide was later identified by immunoblot analysis of nuclear extracts, providing the first evidence that additional AUF1 isoforms exist and could be localized to different subcellular compartments with potentially different biological functions. Subsequent purification and cloning identified AUF1 as a family of four related isoforms derived by alternative splicing of a common pre-mRNA.11 In brief, p37AUF1 isoform lacks both exons 2 and 7, p40AUF1 contains exon 2 but lacks exon 7, p42AUF1 contains exon 7 but lacks exon 2, and p45AUF1 contains the entire AUF1 coding sequence12 (Figure 1(a)). All four AUF1 isoforms contain conserved protein domains including two tandem arranged, nonidentical RNA recognition motifs (RRMs) and an eight-amino acid glutamine-rich motif located C-terminal to the second RRM.11 Although all four isoforms contain two RRM domains, and all have been shown to target ARE-containing mRNAs, they exhibit varied affinities for AREs and different target transcripts. AUF1 isoforms p37 and p42 display threefold to fivefold higher affinity for AU-rich sequences than p40 and p45, thought to be a result of exclusion of exon 7. p37AUF1 has been proposed to have the strongest binding affinity for ARE-containing mRNAs, as it has been found to bind to select target mRNAs in the nanomolar range.13 Additionally, the different functions of the AUF1 isoforms have been proposed to result in part from their subcellular, nuclear, or cytoplasmic localization, most likely in part from the inclusion of exon 2 or 7. In most cells, p37AUF1 and p40AUF1 shuttle efficiently between the nucleus and cytoplasm, whereas p42AUF1 and p45AUF1 isoforms have been shown to shuttle more weakly and are therefore more restricted to the nucleus14–17 (Figure 1(b)). The localization of AUF1 isoforms can be influenced by a variety of factors including the presence or absence of exons containing nuclear localization signals, interaction with transporters or chaperones, protein ubiquitination, and phosphorylation of specific residues.14,18–25



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RNA-binding protein AUF1

(a) Exon composition and cellular localization of AUF1 p37 p40 p42 p45 Exon 2 RRM 1 RRM 2

Q

Exon 7

(b) p37

p42

p40 p45

Nucleus

Cytoplasm

FIGURE 1 | Domain organization and subcellular localization of AUF1 family isoforms. (a) The locations of peptide sequences encoded by alternatively spliced exons 2 and 7 are shown, as are the glutamine-rich (Q-rich) domain and RNA recognition motifs (RRMs) that are found in all AUF1 isoforms. (b) The smaller p37 and p40 isoforms shuttle more actively between the nucleus and cytoplasm compared with the larger p42 and p45 isoforms.

MECHANISM FOR CONTROL OF mRNA FATE BY AUF1 AREs are one of the most well-studied cis-acting elements responsible for changes in mRNA stability. For reasons that remain unclear, AREs only control mRNA decay and/or translation when located within the 3′ -UTR, where they are found in up to 16% of all protein-coding mRNAs.26 The functional ARE is most often composed of copies of an AUUUA sequence motif that can be organized into several classes based on the context of the AUUUA pentamer, the number of repeated motifs, and whether they are present contiguously or not. Inflammatory mediators, such as chemokines and cytokines, generally have multiple contiguous repeats of the AUUUA motif, whereas immediate early response genes such as proto-oncogenes typically contain scattered repeats of the AUUUA pentamer throughout the 3′ -UTR.3,26 AREs are thought to promote mRNA stability or decay by interaction with trans-acting RNA-binding proteins that dictate the fate of the ARE-mRNA.2 AUBPs have not been found to have enzymatic activities in their own right and therefore must serve to recruit decay machineries. Despite the identification of AREs and many of their associated AUBPs more than 20 years ago, there is still no established mechanism by which AREs promote mRNA decay through interaction with AUBPs. There is a multimechanism complexity to mRNA decay, which further obscures the mechanism by which AUBPs function. For instance, Volume 5, July/August 2014

although AUF1 does not contain protein domains capable of ribonucleolytic activity, it is thought to control mRNA fate by recruitment of specific components of the mRNA decay machinery.27 That said, in mammalian cells, mRNA decay is initiated in several ways. For many mRNAs, decay first involves deadenylation of the poly(A) tail, followed by removal of the 5′ 7-methylguanosine cap by decapping enzymes, ultimately culminating in 5′ → 3′ exonucleolytic decay. However, in other cases, mRNAs can be degraded in a 3′ → 5′ fashion through a complex of exonucleases known as the exosome.2 Furthermore, mRNAs can also be targeted for decay by being shuttled to discrete cytoplasmic foci such as processing bodies (P-bodies) or stress granules, which contain multiple factors involved in either mRNA decay or translational repression, respectively.2,28 There are multiple ways in which the AUBP–ARE interaction could potentially promote ARE-mRNA decay or translation inhibition, and all have been invoked as a mechanism of action. Moreover, in the case of AUF1, this protein has been shown to form a complex with translation initiation factor eIF4G, a member of the cap-dependent translation initiation complex, along with heat shock proteins.29 This complex in turn attracts the mRNA degradation machinery, referred to as the AUF1- and signal transduction-regulated complex (ASTRC).30 In so doing, there is evidence that AUF1 protein oligomers are formed following dimerization of AUF1 on ARE-containing mRNAs. AUF1 oligomerization on the ARE is thought to initiate recruitment and assembly of other cellular factors including eIF4G, polyA-binding protein (PABP), hsp70, hsp27, hsc70, and LDH.29–32 How this is related to other potential mechanisms of mRNA decay is unknown. For instance, ubiquitination of p37AUF1 and p40AUF1 , and their degradation by proteasomes, has also been shown to be associated with enhanced ARE-mRNA degradation, possibly through recruitment of exosomes.32,33 Given the absence of physiologically representative cell-free regulated decay systems for AUF1-mediated ARE-mRNA decay, there is currently little direct evidence to substantiate potential molecular mechanisms. It is also possible that AUF1-mediated mRNA decay may occur by any of several mechanisms dictated by the composition of the ARE and the binding of other RNA-binding proteins; none of which can be physiologically tested at the present time. In this regard, mounting evidence suggests that AUF1 can also interact with several other AUBPs involved in modulating ARE-mRNA turnover. It is not understood how AUBPs that seemingly all bind the same


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AREs, possibly simultaneously, can promote different mRNA fates through interaction with the same sequence elements. Thus, the interplay between stabilizing and destabilizing AUBPs may add another level of regulation to ARE-mRNA fate. It seems clear that the fate of an AUBP-bound mRNA is determined by multiple factors, including target ARE sequence (contiguous, interspersed, number of AREs, and flanking sequences), external stimuli, and the relative abundance and subcellular localization of AUBPs. In this regard, the regulatory functions of AUF1 can be influenced by interaction with, or competition for binding to the ARE by different AUBPs. For example, the mRNA-stabilizing protein Hu antigen R (HuR) exerts an opposite influence on the stability of its target ARE-mRNAs than AUF1, stabilizing these mRNAs, despite the fact that these two AUBPs bind common target transcripts, and may compete for the same ARE-binding elements.24 Even more puzzling, HuR and AUF1 have been shown to co-localize on AREs through an interaction that is most likely RNA-dependent.24,34 Thus, HuR and AUF1 may co-occupy the same mRNA by binding adjacent, or possibly the same AREs, perhaps acting as a binary switch controlling ARE-mRNA fate. The intracellular balance of AUF1 and the translation suppression factor TIA-1 related T-cell-restricted intracellular antigen-R (TIAR) is another example. TIAR has also been shown to control target mRNA fate, proposed to be due to competition of ARE binding with AUF1 or other decay factors, and can also be controlled by protein modification through phosphorylation or ubiquitination.35 AUF1 function can also be influenced by interaction with another AUBP mRNA decay-promoting factor, the protein tristetroprolin (TTP). TTP, which promotes ARE-mRNA decay in its own right, has also been shown to enhance the binding affinity of AUF1 for target ARE-mRNAs.36 This raises the possibility that these interacting proteins may function as co-activators, functioning sequentially or simultaneously under different physiologically directed circumstances to regulate ARE-mRNA decay.36 In addition to promoting ARE-mRNA decay, AUF1 has been shown to regulate gene expression at the levels of transcription and translation in certain cellular and tissue contexts. Studies implicate the p42 and p45 AUF1 isoforms in transcriptional activation and translational repression, though there is evidence for occasional involvement of the smaller isoforms as well. Thus, as a family of proteins, AUF1 isoforms have been shown to stimulate ARE-mRNA decay, transcription of certain genes, translational repression, and stabilization of certain ARE-mRNAs.6,35,37–39 552

These specific examples occur in different physiological settings, which will be further discussed below, and are summarized in Table 1.

PHYSIOLOGICAL IMPLICATIONS OF AUF1 DEFICIENCY Shortly after its discovery, it was quickly recognized that AUF1 plays an integral role as a central regulator of multiple biological pathways. This was confirmed with the development of the AUF1 knockout (KO) mouse. Although AUF1-mediated regulation of cellular physiology could be appreciated from previous tissue culture and tissue explant studies, it was the development of the AUF1 KO mouse that has begun to reveal the extraordinary complexity of AUF1-dependent physiological functions. As described in detail below, with AUF1 deficiency, mice display multiple complex phenotypes, many of them tissue, cell type, or context specific. Many, but by no means all the physiological defects are due to loss of regulation of target mRNA degradation. In many cases, the phenotypes are complex because of different AUF1 isoform functions in a specific tissue background, and because the dysregulated mRNAs are involved in multiple different functions. Figure 2 overviews the physiological consequences due to loss of AUF1-mediated mRNA or gene regulation, as characterized in AUF1-deficient mice. It was not surprising that many of the ARE-mRNAs identified to be dysregulated in the AUF1 KO mouse encode cytokines, chemokines, growth factors, and other inflammatory mediators, as this had already been established in tissue culture cells. However, what was surprising is how integral AUF1 is to regulation of inflammation, particularly the regulation of the inflammatory response to bacterial infection, as shown in the AUF1 KO mouse. Proper control of the inflammatory response involves the rapid degradation of ARE-containing mRNAs encoding proinflammatory and anti-inflammatory cytokines, primarily mediated through their interaction with various AUBPs. When challenged with endotoxin, the inflammatory lipopolysaccharide (LPS) outer membrane of Gram-negative bacteria, AUF1 KO mice display symptoms of severe septic shock (endotoxemia), characterized by the uncontrolled expression of proinflammatory cytokines and failure to translate anti-inflammatory cytokine ARE-mRNAs such as that encoding interleukin (IL)-10. AUF1-dependent dysregulation of this process culminates in an inability to attenuate the inflammatory response, eventually concluding in a cytokine storm, catastrophic organ failure, and death.



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TABLE 1 Targets of AUF1 Activity Gene

Physiology

Mechanism of Action

Specific Isoform(s) Implicated

GRO𝛼

Inflammation and cancer

Decay

GM-CSF


Decay

p40AUF1 AUF1

40

IL-1𝛽

Inflammation

Decay

p40

IL-10


Decay

p40AUF1

Decay

p37

AUF1

p40

AUF1

p40

AUF1

IL-6


MCL1

Inflammation

Citation

Decay

41,42 15,25,40 43–45 AUF1

43,46

and p42

, p42AUF1 , and p45AUF1

47 15,25,43,42

TNF-𝛼


Decay

VEGF

Inflammation

Decay

48

IL-2

Inflammation

Decay

49

iNOS

Inflammation

Decay

p37AUF1 , p40AUF1 , p42AUF1 , and p45AUF1 AUF1

50 37

COX-2

Inflammation

Stability

CDKN2A

Cancer

Decay

51

c-Myc

Cancer

Promotion of translation

35,42

Cyclin D1

Cancer

Decay

Dicer1

Cancer

Decay

E2F1

Cancer

Decay

p42

p45AUF1

52

𝛽-AR

Cardiac

Decay

p37

B56𝛼

Cardiac

Decay

p37AUF1

Kv4.3

Cardiac

Decay

SERCA2A

Cardiac

Decay

ENK

Neural development

Transcription

HCV

Virus

Translation

42,52,53 54

AUF1

55 56 57 58

AUF1

p37

AUF1

, p40

AUF1

, and p42

38 59

AUF1

Decay and stability

A1

Spleen development

Stability

39

BCL

Spleen development

Stability

39

p16Ink4

Aging

Decay

ARF

Aging

, and p45

39,47

Spleen development and inflammation

p21

, p42

AUF1

BCL2

cip

p40

AUF1

p37AUF1 and p40AUF1 AUF1

Decay

p37

AUF1

6

AUF1

6

and p40

p19

Aging

Decay

p37

TERT

Aging and cancer

Transcription

p42AUF1 and p45AUF1

This was shown to be the result of homozygous loss of AUF1 and an inability to attenuate inflammatory cytokine expression through its inducible role of enhanced ARE-mRNA destabilization. Endotoxemia, as a result of loss of AUF1, is a consequence of continued expression of tumor necrosis factor (TNF)-𝛼 and IL-1𝛽, among other inflammatory cytokines following immune cell activation, indicating AUF1 is an essential attenuator of the inflammatory response.25 In fact, heterozygous AUF1 KO mice display chronic endotoxemia (but not death) as a result of endotoxin challenge, indicating gene dose importance in AUF1 function and haploinsufficiency. Thus, mutations or polymorphisms that can impair only one copy of AUF1 are sufficient to result in disease. The Volume 5, July/August 2014

6,51,60

AUF1

and p40

6,61

physiological consequences of loss of AUF1 in different tissue backgrounds have further established its essential role in inflammation. AUF1 KO mice have also been shown to develop chronic ectopic dermatitis similar to adult onset psoriasis.49 This is characterized by enhanced dermal infiltration of inflammatory cells, deficient epidermal wound healing responses, and elevated serum IgE levels. Inflammatory cells from AUF1 KO mice, including T-cells and macrophages, display elevated expression of multiple inflammatory mediators, including IL-2, TNF-𝛼, and IL-1𝛽, owing to the inability of AUF1 to rapidly degrade these mRNAs following immune cell activation.49 AUF1 can regulate the expression of many ARE-mRNAs that function in a variety of different


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Diseases of the AUF1 knockout mouse Exon 3 AUF1 locus Targeting vector Neo

TK

AUF1 knockout mouse

m7G

TNF-α

AAAn

m7G

Bcl-2

AAAn

m7G

TNF-α

AAAn

m7G

p16ink4a

AAAn

m7G

IL-1β

AAAn

m7G

A1

AAAn

m7G

IL-1β

AAAn

m7G

p19Arf

AAAn

m7G

Bcl-XL

AAAn

m7G

IL-2

AAAn

m7G

p21CIP

AAAn

mTERT mTERC Control of systemic inflammatory response

Splenic follicular B-cell maintenance

Pruitic skin inflammation and atopic dermatitis

Multifaceted control of aging and cellular senescence

FIGURE 2 | AUF1 knockout mouse displays multiple physiological abnormalities. The AUF1 knockout mouse was generated by homologous recombination in mouse embryonic stem cells by targeting the RNA-binding motif containing the third exon of AUF1, allowing for disruption of the remainder of the reading frame. Neo, neomycin-resistant cassette; TK, thymidine kinase cassette; filled box, coding region; open box, noncoding region. Loss of AUF1 results in dysregulation of multiple mRNA targets and transcription of select genes leading to specific phenotypes observed in the AUF1 knockout mouse.

physiological pathways. Therefore, AUF1 is a key regulator of various physiological responses with complex immunological manifestations. For example, development of B-lymphocyte subsets in the spleen and other lymphoid organs is essential for proper immune function. B-lymphocyte deficiencies are associated with opportunistic bacterial infection, in part resulting from deficient antibody-secreting plasma cells. AUF1 deficiency was shown to lead to reduced spleen size owing to a significant reduction in the formation of splenic B-lymphocyte germinal centers, sites of follicular B-lymphocyte development.39 Loss of AUF1 expression results in reduced expression of essential mediators of B-lymphocyte death including Bcl-2, A1, and Bcl-XL , thereby increasing apoptosis of mature follicular B lymphocytes, the major mature B-cell population in the spleen that develops into IgG antibody-secreting plasma cells.39 Moreover, loss of AUF1 was also associated with the translation blockade of another AUF1 target ARE-mRNA, 554

lymphotoxin-𝛼, similar to the mechanism by which AUF1 regulates IL-10, which is involved in the formation of germinal centers for B-cell development. Thus, it has become apparent that AUF1 not only promotes normal physiological regulation of ARE-mRNA stability but, in some cases, also the translational control of ARE-mRNAs with opposing function. Although not well studied, it is suspected that loss of AUF1 allows binding of translation-inhibiting AUBPs to certain ARE-mRNAs, such as TIA-1 or TIAR. Why certain ARE-containing mRNAs may be targets for opposing regulation by translation repression and others are not known, particularly with little difference in ARE context or sequence, and is an area of study that remains unresolved. The high complexity of AUF1 function is manifested in its physiological role in telomere maintenance. This was highlighted by the discovery of simultaneous premature aging, tissue senescence, inflammation, and increased incidence of cancer



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phenotypes in AUF1-deficient mice. Although AUF1 is an established attenuator of inflammation, and inflammation is associated with DNA damage and cellular senescence, this alone could not explain the complex premature aging phenotype of AUF1 KO mice. To this end, AUF1 was shown to posses several independent functions. The two smaller AUF1 proteins were shown to more efficiently promote the destabilization of the cell cycle checkpoint regulator gene mRNAs, p16ink4 , p21cip , and p19ARF .6 Loss of p37AUF1 and p40AUF1 therefore results in cellular senescence and widespread tissue atrophy. The two larger AUF1 isoforms were found to bind and activate the promoter of the telomerase catalytic subunit Tert, thereby promoting the expression of Tert mRNA and strongly increasing its protein levels. AUF1 KO mice therefore display telomere erosion, increased DNA damage responses at telomere ends, enhanced cellular senescence, and premature aging, all of which increase with each generation, a process known as genetic anticipation.6 It is clear that AUF1 deficiency has severe and complex consequences that manifest as a number of diseases and syndromes found in humans. AUF1 isoforms are differentially expressed in different tissue types, indicating that AUF1 is an important gene expression regulator whose targets will be different in different cellular contexts. The pattern of expression of AUF1 in different tissues is similar between mouse and human, indicating that there is every reason to believe that physiological pathologies discovered in mouse resulting from AUF1 deficiency are likely representative of human disease. Tissue and isoform distribution patterns of AUF1 are summarized in Figure 3.

Inflammation Some of the first evidence supporting a role for AUF1 in inflammation was the discovery that AUF1 controls the stability of the granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE-mRNA. GM-CSF stimulates myeloid proliferation, differentiation, and activation, and provokes the septic shock response when overexpressed. Activated mononuclear cells were shown to display decreased expression and shorter half-life of GM-CSF mRNA compared with nonactivated cells, because of increased expression of AUF1 and increased binding of AUF1 to the GM-CSF ARE-containing 3′ -UTR.41 Shortly after this novel finding, AUF1 was identified as a key binding factor for the 3′ -UTR of GRO𝛼 and IL-1𝛽 mRNAs that is critical for the regulation of these transcripts during monocyte adherence.40 Post-translational modifications, subcellular localization, and differential expression levels of the Volume 5, July/August 2014

four AUF1 isoforms all impact AUF1-mediated regulation of target mRNAs. The role of post-translational modifications on AUF1 activity was examined in the context of inflammation. p40AUF1 was found to be selectively phosphorylated at Ser83 and Ser87 in monocytic leukemia cells. Phosphorylation at these sites was associated with rapid degradation of IL-1𝛽 and TNF-𝛼 mRNAs, indicating that specific AUF1 isoforms may control the turnover of specific target mRNAs in a manner dictated by regulated, upstream signal transduction pathways, including phosphorylation and possibly other post-translational modifications.15 Differential expression of AUF1 isoforms is likely a method by which target mRNA stability and protein expression levels are physiologically regulated, including proper control of the inflammatory response. For example, increased expression of AUF1 has been shown to be a contributing factor during the initial phases of type I diabetes, which is characterized by a cytokine-mediated inflammatory reaction leading to progressive loss of pancreatic 𝛽 cells. In particular, overexpression of the p40AUF1 , p42AUF1 , and p45AUF1 isoforms mimics the effect of cytokine-induced apoptosis of pancreatic 𝛽 cells, an effect shown to be due in part to AUF1-mediated regulation of the antiapoptotic genes Bcl2 and Mcl1.47 Additionally, AUF1 has been shown to regulate expression levels of inducible nitric oxide synthase (iNOS), which is responsible for the production of nitric oxide in the innate immune system, a crucial part of the defense against invading microorganisms. All four AUF1 isoforms were found to bind the 3′ -UTR of the iNOS ARE-mRNA and to promote its rapid decay and decreased expression.50 Taken together, these studies implicate differential expression levels of AUF1 isoforms as key regulators of the immune response. Nuclear–cytoplasmic localization of AUF1 is also a key control point for AUF1-mediated regulation of target mRNA expression and stability, as the cytoplasm is the predominant site of mRNA degradation and translation. Following LPS stimulation, AUF1 has been shown to translocate from the nucleus to the cytoplasm, dependent on the presence of the protein MKP-1, a negative regulator of the host inflammatory response following infection.43 MKP-1-dependent translocation of AUF1 is associated with enhanced degradation of IL-6, IL-10, and TNF-𝛼 mRNAs, which are in turn associated with increased AUF1 binding to these transcripts.43 Research on human synovial fibroblasts has further established the importance of AUF1 subcellular localization in controlling inflammation. COX-2 is the rate-limiting enzyme responsible for proinflammatory


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Tissue distribution and physiological implications of altered AUF1 expression Telomere maintenance mTERT aging

Neuronal development ENK

Thyroid cancer Epstein–Barr virus

Lung cancer Breast cancer p16INK4A

Chronic dermatitis IL-2

Cardiac maintenance Kv4.3, B56α, SERCA2A Myocardial infarction B-AR

Inflammation TNF-α, IL-1β, GM-CSF, Gro-a, IL-6, COX2, VEGF, sFlt-1 B-cell development Bcl-2, A1, BCL-XL

Liver cancer Hepatitis C HCV

Type 1 diabetes BCL2, MCL1, iNOS

Sarcoma c-Myc, c-jun, c-fos, cyclin D1

Lymphoma HIV

Melanoma IL-10

FIGURE 3 | Tissue and isoform distribution of AUF1 expression. AUF1 analysis in mouse and human tissue specimens demonstrates tissue- and isoform-specific distribution in a wide range of organs. In mice, AUF1 is highly expressed in the spleen and thymus, moderately expressed in the brain, reproductive organs, heart, liver, kidney, and skeletal muscle, and displays faint expression in the intestine and lung. In the brain, testis, and uterus, p45AUF1 , p42AUF1 , and p40AUF1 isoforms are predominantly expressed compared with the p37 isoform. In contrast, p37AUF1 , p40AUF1 , and p42AUF1 isoforms are principally expressed in the lung and ovary where p45AUF1 is undetectable. Similar expression levels of AUF1 are found in human tissue specimens. However, AUF1 expression may be more ubiquitous than in the mouse, as AUF1 can be detected in all human tissues. AUF1-positive cells were detected in all human tissues, possibly increasing with age in the immune system.

prostaglandin synthesis. Cytoplasmic p42AUF1 was found to stabilize the COX-2 ARE-mRNA by specifically binding the COX-2 mRNA 3′ -UTR, leading to increased COX-2 mRNA stability and increased protein expression.37 As described earlier, AUF1 has also been implicated in the inflammatory response to bacterial endotoxin (LPS). Exposure of monocytes and macrophages to endotoxin leads to the production of proinflammatory cytokines, followed by production of anti-inflammatory IL-10 that attenuates the immune response. AUF1 has been shown to play a role in regulating both the proinflammatory and anti-inflammatory responses. LPS typically mediates a proinflammatory cytokine response, followed by induction of IL-10 gene expression dependent on p40AUF1 , which promotes IL-10 mRNA stability 556

and translation.44 This is in contrast to the absence of AUF1 in which IL-10 mRNA is translationally repressed. AUF1 therefore has many functions in regulating the immune response. Yet one more function comes from evidence that AUF1 regulates the stability of certain inflammation-associated mRNAs through interaction with mature miRNAs. miR-221 expression is increased following LPS stimulation, and recruits AUF1 to the TNF-𝛼 mRNA 3′ -UTR, orchestrating attenuation of the immune response by further enhancing degradation of the TNF-𝛼 transcript.62 AUF1 is also involved in regulating angiogenesis during inflammatory wound healing. During severe inflammation there is considerable tissue remodeling, in which macrophages produce a variety of inflammatory mediators, including vascular endothelial growth factor (VEGF), which is critical for angiogenesis,



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vascular permeability, and stimulation of proangiogenic immune cells. AUF1 was found to attenuate VEGF expression through interaction with the ARE-containing 3′ -UTR of VEGF mRNA, promoting accelerated mRNA decay. Thus, AUF1 is a key regulator and coordinator of VEGF expression, angiogenesis, and proper wound healing responses.63 Taken together, these studies show that the AUF1 family of proteins orchestrates a multifaceted regulatory program during an inflammatory response at the levels of mRNA transcription, stability, and translation.

Cancer It has been well documented that expression of AUBPs and their intracellular localization are often dysregulated in human cancer and associated with aberrant protransforming gene regulation. Altered AUF1 expression has been implicated as a contributing factor in the development and progression of cancer in a variety of tissues. The role of AUF1 in cancer has been reviewed in detail by Zucconi and Wilson, and will therefore only be briefly discussed here. Readers are encouraged to read this review for a more comprehensive examination of AUF1 in cancer.64 Some of the first evidence that altered AUF1 expression may contribute to neoplastic transformation was the observation that transgenic mice engineered to globally overexpress the p37AUF1 isoform develop spontaneous soft tissue sarcomas.63 These mice display dysregulated overabundance of multiple cancer-associated mRNAs, including c-Myc, c-jun, c-fos, GM-CSF, TNF-𝛼, and cyclin D1.42 All of these ARE-mRNAs are established targets of AUF1. Unanswered, however, is how overexpression of the p37AUF1 isoform, the isoform most associated with increased decay of proto-oncogene ARE-mRNAs, could instead result in their increased abundance. Nevertheless, these findings provided the first indication that abnormal AUF1 expression is associated in some manner with cancer development in experimental animal models. In a large-scale cancer mRNA profiling array, mRNA levels of AUF1, TIA-1, TTP, and HuR were compared between tumors and adjacent normal tissues in tumors of 154 patients representing 19 different cancer etiologies. AUF1 expression was shown to be significantly increased in 13% of tumors, consistent with other reports that found AUF1 to be upregulated in breast, skin, thyroid, and liver carcinomas.64–67 Unfortunately, given the very limited RNA and protein sequence differences between the four AUF1 isoforms, it was not possible to determine which of the different isoforms are differentially expressed (overexpressed or underexpressed) in association with development of different human cancers. Volume 5, July/August 2014

It is evident that changes in AUF1 expression can lead to dysregulated control of target ARE-mRNAs, and many groups have attempted to elucidate the complex mechanisms underlying changes in ARE-mRNAs observed during neoplastic transformation. AUF1 expression has been shown to be under the control of p16ink4a , the same cyclin-dependent kinase inhibitor whose mRNA is a target of AUF1, and plays important roles in tumor suppression and cell cycle inhibition. p16ink4a expression has been found to be lost in a variety of cancers through several mechanisms, thereby relieving cell cycle checkpoint control.68 Attenuation of p16ink4a expression is associated with increased nuclear and cytoplasmic levels of AUF1, consistent with AUF1-mediated decay of its mRNA.69 Reduced levels of p16ink4a are also associated with reduced levels of other AUF1 target mRNAs, including transcription factor E2F1 and cyclin D1, thereby leading to decreased levels of apoptosis in addition to increased cell proliferation.52 Other studies also showed that AUF1 influences p16ink4a expression, particularly in breast cancer-associated fibroblasts (CAFs), consistent with the observation that AUF1 is upregulated in CAFs compared with histologically normal tissue.51 Increased expression of AUF1 was also found to be responsible for reduced stability and expression of CDKN2A (cyclin-dependent kinase 2) mRNA levels in CAFs, again indirectly resulting in decreased p16ink4a expression. Thus, AUF1 is a master regulator of a complex network of genes, which when abnormally expressed, can participate in cancer development and progression.51 The intracellular localization of AUF1 can also contribute to altered and protransforming gene expression in neoplastic tissues. Subcellular localization of AUF1 therefore appears to be an important factor in controlling the expression of multiple cancer-associated mRNAs. For example, AUF1 regulates IL-10 expression in melanoma as it does in the context of inflammation. As an anti-inflammatory factor, IL-10 is a key player involved in suppressing immune surveillance and tumor rejection. Like studies in immune and epithelial cells, AUF1 induces rapid decay of the IL-10 mRNA in normal melanocytes. However, in malignant melanoma cells there is increased transcription and stability of IL-10 mRNA,45 which was shown to be due in part to the increased cytoplasmic localization of AUF1 in normal melanocytes where it binds and degrades the IL-10 mRNA compared with decreased localization in melanoma cells.45 A pattern of increased cytoplasmic expression of AUF1 has been shown to be correlated with hyperplasia and neoplasia of cancers other than melanoma,


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as shown in mouse lung tissue and in human thyroid tissues.70 In thyroid cancer, cytoplasmic accumulation of AUF1 was found to be strongly increased in malignant cells compared with normal thyroid tissue. Increased cytoplasmic expression of AUF1 was also associated with increased proliferation and transformation of thyroid cells, in concert with the reduced abundance of key cell cycle checkpoint regulators, including p16ink4a , an established target of AUF1.60 Considering that AUF1 expression and localization have been consistently shown to be dysregulated in many different cancer types, it is understandable that the stability or translation of AUF1 target mRNAs, some of them pro-oncogenic, is also altered in response to changes in AUF1 expression or localization. In this regard, several early conflicting issues have now been resolved. For instance, AUF1 was originally implicated in promoting decay of the c-Myc mRNA,8 which should be antioncogenic, yet c-Myc has been shown to be overexpressed in many human tumors where it is a major contributing factor to cancer initiation and progression.8 However, AUF1 is also overexpressed in many human cancers. Later studies established that AUF1 does not in fact strongly target the c-Myc mRNA for accelerated decay, but instead largely promotes its translation.35 Further investigation into AUF1-dependent regulation of c-Myc expression demonstrated that its mRNA simultaneously associates with both AUF1 and ING4, a protein that plays several distinct roles in cancer-related cellular processes, one of which is to increase c-Myc mRNA translation. Thus, these results indicate that cytoplasmic AUF1 is a key mediator of increased c-Myc expression, which can be altered in different cellular and intracellular contexts.71 Another mechanism by which AUF1 can affect gene expression, and suspected to be involved in an AUF1 role in cancer development, is through alteration of miRNA expression. RNA-binding proteins have been shown in multiple studies to interact with miRNAs and influence their ability to regulate target mRNA stability or translation.72–74 Expression of AUF1 and DICER1, a key nuclease in miRNA biogenesis, has been shown to be inversely correlated in select tumor types. AUF1 was shown to attenuate DICER1 expression by promoting rapid decay of its mRNA, leading to decreased levels of specific miRNAs and reduced repression of their target mRNAs.75 AUF1 may therefore play a regulatory role in miRNA biogenesis, which commonly occurs in many cancers.75 Investigation into the physiological functions of the different AUF1 isoforms in cancer has further confirmed distinct roles for the different AUF1 isoforms in different cellular and tissue contexts. In contrast to 558

the cancer-promoting characteristics of p37AUF1 overexpression in AUF1 transgenic mice, prostaglandin A2 induces expression of the p45AUF1 isoform in non-small-cell lung cancer cells through an unknown mechanism. This suggests that prostaglandin A2 has an antiproliferative role owing to increased p45AUF1 expression and its degradation of cyclin D1 mRNA.53 Additionally, knockdown of AUF1 has been shown to stabilize IL-6 mRNA, and elevated expression of IL-6 protein is associated with increased tumor growth.69 Interestingly, the IL-6 mRNA specifically co-immunoprecipitates with the p37AUF1 and p42AUF1 AUF1 isoforms in cell extracts, further indicating that there are specific roles for the different AUF1 isoforms in regulating gene expression.46 Enhanced or inappropriate activation of telomerase is yet another hallmark of tumorigenesis, allowing cancer cells to evade programmed cell death and proliferate continuously. As described earlier, studies from the AUF1 KO mouse revealed an integral role for AUF1 in regulating several aging-associated pathways, including regulation of telomerase expression. Loss of AUF1 was found to result in severely reduced telomere length in proliferative cells owing to loss of AUF1-dependent upregulation of telomerase enzyme (TERT) mRNA levels thought to be involved in the activation of Tert transcription.6 Reduced telomere length as a result of AUF1 deficiency results in increased chromosomal abnormalities, rearrangements,6 and emergence of multiple tissue neoplasms in the mouse (unpublished results). The role of AUF1 in regulating telomerase activity was further validated in human oral squamous carcinoma cells, as knockdown of AUF1 also reduced TERT promoter activity in this system.61

Cardiac Homeostasis Surprisingly, many mRNAs involved in cardiovascular homeostasis are also established targets of ARE-mediated mRNA regulation by transacting AUBPs.76 A central feature of heart disease is the molecular remodeling of signaling pathways in cardiac myocytes. AUF1 expression has been shown to be altered in response to these signaling pathways and in turn may control the stability of many mRNAs that contain AREs and are involved in cardiovascular function. Mutations, polymorphisms, or changes in AUF1 isoform levels may therefore be involved in certain cardiovascular diseases, an intriguing but as of yet unproven possibility. For example, the outward current Kv4 channels in human myocardium are often downregulated during cardiac hypertrophy, which is a pathological thickening of the myocardium as a result of hypertension.77



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Treatment of cardiac myocytes with angiotensin II, a key blood pressure regulator, can recapitulate the downregulation of Kv4 channels. Importantly, treatment with angiotensin II also upregulates AUF1 through AT(1), NADPH oxidase, and p38 MAP kinase signaling pathways, promoting AUF1-mediated rapid decay of the Kv4.3 mRNA. Therefore, treatment with angiotensin II not only increases AUF1 expression but also facilitates enhanced binding of AUF1 to the Kv4.3 ARE-mRNA 3′ -UTR, thereby leading to decreased mRNA stability and reduced protein expression.57 AUF1 has also been shown to be upregulated in response to sustained mitogen-activated protein kinase 8 (MAPK8, JNK) activation in cultured cardiac cells. The regulated balance between kinases and phosphatases is essential for normal cardiomyocyte function as in other cells, and changes in the expression of the catalytic subunit of phosphatase PP2A have been shown to contribute to stress-activated contractile function of cardiac myocytes during hypertrophy.78 JNK, a member of the stress-activated MAP kinase family, and its upregulation of AUF1, was shown to lead to enhanced mRNA degradation and reduced expression of B56𝛼 protein, a targeting subunit of the phosphatase PP2A.56 Thus, AUF1 in part plays a role in cardiac function by regulating the stability of a major phosphatase in response to its stress-regulated phosphorylation. There are a variety of other cardiac function mRNAs that are also targets of AUF1. AUF1 has been found to bind the 3′ -UTR of the SERCA2A mRNA, a calcium-regulated ATPase commonly downregulated in the sarcoplasmic reticulum of cardiomyocytes in patients with cardiac hypertrophy and heart failure. In addition to other signaling pathways that can mediate AUF1 function, AUF1 displays increased binding affinity for the SERCA2A 3′ -UTR in response to protein kinase C (PKC) activation. The importance of post-translational modification and localization of AUF1 was also evident in this study, as AUF1 binding to the SERCA2A mRNA 3′ -UTR was primarily observed in the nucleus, and threonine phosphorylation of AUF1 was increased following PKC activation.58 Possibly, the most striking evidence implicating AUF1 as a key mediator of cardiovascular maintenance is the finding that AUF1 mRNA and protein levels are significantly increased in the ventricular myocardium tissue from patients with myocardial infarction.55 Heart failure is associated with heightened activity of the adrenergic nervous system, and more severe cases are associated with increased circulating and cardiac levels of norepinephrine. Volume 5, July/August 2014

Exposure to norepinephrine, a 𝛽-adrenergic receptor (𝛽-AR) agonist, results in a 100% increase in AUF1 expression with a simultaneous twofold decrease in 𝛽-AR ARE-mRNA abundance. Consistent with other reports, p37AUF1 was found to be expressed and localized to the polysome fraction and to bind the 𝛽-AR mRNA 3′ -UTR. AUF1 binding to the 𝛽-AR mRNA was further found to promote its destabilization, confirming unique roles for the different AUF1 isoforms.55 Mitochondrial proteins have been shown to contribute to the divergent mitochondrial densities observed in striated muscle that affect muscle oxidative capacity, an important component of cardiac homeostasis. The mitochondrial transcription factor and ARE-mRNA-encoded Tfam, PGC-1a, and NRF-2a were shown to be rapidly degraded in cardiac and slow-twitch red muscle compared with fast-twitch white fibers. Similarly, AUF1 was shown to be elevated in cardiac and slow-twitch muscle when compared with fast-twitch fibers, potentially contributing to the observed reduced levels of mRNA stability for these mitochondrial genes. Interestingly, the RNA-binding protein HuR was elevated to a similar extent as AUF1 isoforms p42AUF1 and p45AUF1 in cardiac muscle cells, whereas the p37AUF1 and p40AUF1 isoforms were expressed at lower levels, as they are all in skeletal muscle cell types.79 Thus, increased expression of the two smaller AUF1 isoforms is consistent with functionally decreased stability of mitochondrial mRNAs involved in oxidative capacity. These data indicate that alterations in specific AUF1 isoform expression levels are associated with hypertrophic heart disease. Moreover, they provide supporting evidence that changes in AUF1-mediated mRNA stability, AUF1 phosphorylation, and its intracellular distribution are all important mechanisms that can control changes in gene expression evoked by stress-activated pathways in cardiac cells.

Viral Infection and Replication Following viral infection, host RNA-binding proteins are often hijacked by viruses and repurposed to function in viral mRNA macromolecular functions, including translation and viral genome replication. AUF1 has been shown to interact with a number of viral proteins, seemingly to ensure a competent viral replication cycle. AUF1 has been shown to interact with the internal ribosomal entry site (IRES) in the 5′ -UTR of the hepatitis C virus (HCV) mRNA, one of the major causative agents of liver cirrhosis and hepatocellular carcinoma. Through its interaction with the HCV 5′ -UTR, AUF1 apparently promotes


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HCV infection via increased translation of the HCV mRNA.59 The specific details of the mechanism of action are unknown. Additionally, all four AUF1 isoforms were found to be cleaved by a picornaviral protease encoded by poliovirus and human rhinoviruses during infection, which is associated with the increased nuclear to cytoplasmic shuttling of AUF1.80 The biological consequences of AUF1 proteolysis have not been established. AUF1 has also been shown to interact with the EBER1 RNA, a noncoding RNA expressed by Epstein–Barr virus that might prevent the antiviral type 1 interferon response. EBER1 can compete with p40AUF1 for binding to ARE-containing mRNA targets, suggesting that AUF1 interaction with EBER1 may disrupt the ability of AUF1 to regulate specific mRNA targets.81 Similar to infection with poliovirus and rhinoviruses, HIV infection results in the relocalization of AUF1 from the nucleus to the cytoplasm where it is associated with the HIV Gag protein. Although loss of AUF1 results in accumulation of unspliced HIV-1 RNAs, overexpression of each of the four AUF1 isoforms results in varied effects on Gag expression.82 Taken together, these results indicate that AUF1 is a key accessory protein in virus replication that acts at different levels in different viruses, and involves both differential AUF1 isoform expression levels and subcellular localization.

Neural Development and Function Like all other tissues, coordinated expression of genes is critical to normal neural development and can be influenced by both genetic and epigenetic factors. It has been recently appreciated that post-transcriptional regulation is a key control point in mediating proper neural development, and AUF1 has been implicated as a key factor governing these processes. Most work has focused on isoform-specific expression levels of AUF1 throughout neural development and the impact of these expression patterns on downstream targets. To date, studies have not yet tested an association between AUF1 expression levels and neuronal functions. In a rat model, AUF1 is detected as early as embryonic day 14 (E14) in a subset of neural cell progenitors. By E18, practically all cells in the developing cortex are AUF1 positive. Early embryonic expression of AUF1 indicates that the protein is present in the majority of actively proliferating cells, as these are the most abundant cell type at this developmental stage.83 All four AUF1 isoforms are expressed in the developing brain, with p40AUF1 and p42AUF1 displaying the highest expression levels.38 A complementary study focused on cerebellar formation observed 560

that p37AUF1 , p40AUF1 , and p42AUF1 mRNA and protein levels are largely unchanged throughout cerebellar development, with the exception of p45AUF1 , which is progressively increased.84 Multiple studies also confirm that all AUF1 isoforms are strongly attenuated in adulthood, beginning a steady decline after birth (p2), ultimately expressed at their lowest levels in p28 brain.84 AUF1 is expressed during early cortical neurogenesis and is primarily located in the ventricular and subventricular zones. Through its interaction with HDAC1 and MTA2 proteins, AUF1 is thought to coordinate gene expression at different stages of neuronal lineage progression by recruiting chromatin remodeling molecules to AT-rich DNA elements.83 In an effort to determine the regulatory mechanisms governing enkephalin (ENK) gene expression in the developing brain, AUF1 was also shown to play a role in mediating gene expression at the transcriptional level, a theme in AUF1 function that has emerged in other systems. As described earlier, individual AUF1 isoforms are associated with distinct regulatory roles. For instance, in the brain, only p37AUF1 , p40AUF1 , and p42AUF1 can bind AT-rich dsDNA in order to regulate cell-specific expression of the ENK gene.38 In addition to specific isoform and abundance expression patterns throughout neural development, AUF1 expression is also altered following NMDA receptor stimulation. Activation of NMDA receptors results in a wide range of intracellular cascades essential for neuronal cell survival, differentiation, and neuroplasticity. In cerebellar granule cells, stimulation with NMDA results in attenuation of all AUF1 proteins, resulting in increased levels of the AUF1 target mRNA encoding the 𝛼 2 subunit of guanylyl cyclase, a key factor involved in guanylyl cyclase localization and neuronal cell differentiation.85

CONCLUSION Regulated mRNA decay is an essential mechanism governing changes in inducible gene expression. AREs are cis-acting regulatory elements present within the 3′ -UTR of select mRNAs that can dictate mRNA half-life through association with specific AUBPs that can guide the mRNA for decay, stabilization, or translational suppression.2 AUF1 is an ARE-binding protein that primarily facilitates rapid decay of targeted ARE-mRNAs in a multistep but poorly understood process.12,27 AUF1 is expressed in most or all tissues, although there are wide differences in expression levels and the isoforms expressed.86,87 AUF1 isoforms have been shown to be differentially expressed and modified in different tissues, alterations of which are



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thought to play important roles in certain disease etiologies based on animal models. In some cases, there is strong evidence that changes in AUF1 expression are associated with certain pathophysiologies. Different AUF1 isoforms possess distinctly different regulatory functions that are defined by intracellular location, tissue distribution, and expression context.86 Given its comprehensive targeting ability, AUF1 may act as a coordinator of the activity of interrelated pathways. Evidence for this view is derived from AUF1 deficiency in the KO mouse. AUF1 deficiency in an engineered KO mouse model has highlighted the importance of AUF1 in controlling the inflammatory response at multiple levels and the interaction of this response with cellular senescence, and tissue fate by coordinating the regulation of targeted mRNA decay, cell cycle control, and even gene transcription involved in these physiological responses. While AUF1 is a key regulator of the expression of multiple inflammatory mediators through the stability and translation of their mRNAs, and is thus a central regulator of immune and inflammatory responses, it is also a regulator of the stability or translation of multiple mRNAs involved in cancer initiation and progression. Thus, AUF1 provides a direct link between the control of inflammation and some of the molecular alterations involved in cancer development. Given its fairly pleiotropic and

tissue-specific effects, AUF1 has also been implicated in neuronal development, cardiac homeostasis, and viral replication, among other physiologically relevant processes, all of which assume differential specificities in regulation that differ with tissue type, AUF1 isoform expression levels, and intracellular distribution. This review has summarized the regulatory roles of AUF1, its target mRNAs, genes, and pathways influenced by AUF1, pathophysiologies associated with its altered expression, and the complexity of AUF1 mechanisms of action. By controlling target gene transcription, mRNA translation, and mRNA stability for constellations of genes in different cellular and tissue contexts, AUF1 enacts and coordinates a program of genetic regulation involving multiple genes and biochemical pathways. Looking forward, more work must be done to determine how AUF1 isoform-specific expression in different tissue and disease etiologies produces the different specific regulatory roles these isoforms play, and how their deficiency or overexpression is involved in mediating particular disease states. A more comprehensive understanding of AUF1 isoform-mediated regulation of target genes and their mRNAs may provide the basis for therapeutic intervention in specific pathologies in which AUF1 isoforms play distinct roles.

ACKNOWLEDGMENTS This study was supported by NIH grant GM085693 (RJS) and T32 CA009161 (AM) and T32 GM 13-A0-S1-090476 (DMC) training grants.

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