Regulation of context-specific gene expression by posttranscriptional switches

Abstract

Post-transcriptional control of mRNA is a vital step in the regulation of gene expression. Highly specific combinations of RNA-binding–proteins (RBPs) and microRNAs conjointly contribute to the complexity of post-transcriptional control of mRNAs. Post-transcriptional switches regulated by RBPs control context-specific expression of two alternative gene products from a single transcript.

Keywords: :

• RNA binding proteins
• post-transcriptional switch
• untranslated regions of mRNA
• exonic miRNA
• miRNAs
• wound healing
• non-coding RNAs

In the pathway from transcription to translation, RNA was generally considered as a passive molecule. The structural components of mRNAs usually consist of a 5′- m7GpppN cap, a 5′-untranslated region (UTR), the coding region, a 3′-UTR and a poly(A) tail. Initiation of translation occurs by a sequence of events starting with the binding of a cap-binding complex, recruitment of the pre-initiation complex with 40S ribosomal subunit, scanning and, finally, recruitment of the 60S large ribosomal subunit.¹ Adding to the intricacy, interaction of RNA-binding proteins (RBPs) with 5′- or 3′-UTRs of mRNA and sequence-specific binding of microRNAs (miRNAs), together control the efficiency of translation of the transcript.

Transcripts and cis-Regulatory modules

Transcript-specific translational control of eukaryotic gene expression is often directed by a structural element in the 3′-UTR of the mRNA.² In most vertebrates, 3′-UTRs are substantially longer than their 5′ counterparts, suggesting a significant potential for regulation. Furthermore, a multi-species analysis indicates an increase in the average length of 3′-UTR sequences during evolution, highlighting a correlation of these sequences with increasing complexity of the organism.³ Several protein-coding genes contain miRNA binding sites within their 3′-UTRs. miRNAs are small, non-coding RNAs that influence diverse biological functions through the repression of target genes. It has been proposed that complete complementarity between miRNA and target mRNA leads to the degradation of target mRNA,⁴ whereas partial complementarity may lead to translational repression and/or mRNA degradation.⁵

MicroRNAs can be classified broadly into three categories: intergenic, intronic and exonic, based on genomic location, coordinates, and source of primary transcript.⁶ Although 52% of human miRNAs are intergenic, ~43% of the remaining human miRNAs are encoded within the host genes. One notable feature of intragenic miRNAs is the co-regulation of both miRNAs as well as host mRNA with a single transcription factor/pathway. The remaining ~5% of miRNAs are encoded from the exons of protein coding genes. They are referred to as exonic miRNAs, characterized by the presence of precursor-miRNA stem loop within the exon of the transcripts.⁷ The first exonic miRNA identified was miR-675 derived from the first exon of H19 non-coding RNA.⁸ miR-675 is highly expressed in placenta. In majority of the tissues miR-675 processing from H19 long non-coding RNA is suppressed by an RBP, HuR.

A Repertoire of RBPs Coordinate Multiple Post-transcriptional Events

RBPs interact with regulatory elements of transcripts and control spatial and tissue-specific expression of proteins.³ After transcription, the fate of the transcript is determined by a complex interplay of RBPs and regulatory elements within the transcripts. In multiple instances, RNA stabilizing RBPs like HuR inhibit miRNA mediated repression/deadenylation of target mRNAs by dissociating miRISC/Ago complex from target mRNAs.⁹ HuR promotes translation of nucleolin mRNA in HeLa cells. However, in the absence of HuR, miR-494 translocates nucleolin mRNA to processing bodies (P bodies).¹⁰ Some of the multifunctional RBPs also play a vital role in the biogenesis/processing of miRNAs. Trabucchi et al. detected the association of KH-type splicing regulator protein (KSRP) with the Dicer complex, suggesting a potential role for KSRP in miRNA biogenesis.¹¹ Interestingly, processing of a single miRNA may be controlled by multiple developmentally regulated proteins. Let-7 is regulated by Lin28 in embryonic cells, whereas KSRP promotes the biogenesis of this miRNA in differentiated cells. Paradoxically, another RBP, hnRNPA1, competes with KSRP in binding to the terminal loop of pri-let-7 and inhibits let-7 processing.^12,13 KSRP-mediated miR-155 biogenesis is also inhibited by an RBP Tristetraproline (TTP) in fibrotic lung epithelial cells, although the exact mechanism of this inhibition is unclear.¹⁴ Furthermore, KSRP interacts with both the primary-miRNA transcript and the stem-loop precursor through a G-rich motif present in a subset of mRNAs and promote miRNA biogenesis.¹¹

An Inter-play Between RBPs and Growth Factors Control Post-transcriptional Switches

An intricate network of secreted factors and intracellular signaling intermediates control robust gene expression by cross-talks or direct interaction with RBPs. Growth factors, such as transforming growth factor-β (TGF-β) involved in signaling pathways, modulate the activities of the components of the miRNA biogenesis and indirectly control miRNA expression patterns.¹⁵ The major elements in TGF-β signaling are TGF-β ligands, their receptors, and intracellular Smad effectors. Signal transducers of the TGF-β pathway, the Smad proteins, also modulate miRNA expression by transcriptional and post-transcriptional mechanisms.¹⁶ TGF-β and Bone Morphogenetic Protein 4 (BMP4) signaling stimulate the production of pre-miR-21 by facilitating Drosha-mediated processing. Nuclear accumulation of receptor-Smads (R-Smads) proteins, regulate miRNA maturation by associating with the Drosha/DGCR8 complex.¹⁷

Highlighting some of the above-mentioned events we describe an elegant post-transcriptional switch that controls the context-specific expression of miR-198 or follistatin-like-1 (FSTL1) from a single transcript.¹⁸ miR-198 is a primate-specific miRNA and belongs to a small cohort of human exonic miRNA stem–loops located within the eleventh exon of the protein-coding gene, FSTL1.Cutaneous epidermal keratinocytes express high levels of miR-198. Targeting multiple genes including urokinase-type plasminogen activator (PLAU), diaphanous homolog 1 (DIAPH1), and laminin gamma 2 chain (LAMC2), essential for migration, miR-198 inhibits keratinocyte migration. However, upon skin injury, miR-198 levels plummet as early as 3 hours after wounding, resulting in expression of the target genes and migration of keratinocytes toward the wound edge. Most importantly, expression of miR-198 from the 3′-UTR of FSTL1 mRNA in normal skin switches to expression of the linked open reading frame (ORF) of FSTL1 upon wounding. Expression of glycoprotein FSTL1 was clearly observed at the wound edge, but not in unwounded normal epidermal keratinocytes. Unlike miR-198, FSTL1 facilitates keratinocyte migration. A clear inverse correlation exists between the expression pattern of FSTL1, essential for effective migration and miR-198, an inhibitor of migration.¹⁸ A single transcript can therefore function either as a primary miRNA transcript making miR-198 or function as an mRNA producing FSTL1 protein, suggesting a post-transcriptional switch regulating their context-specific expression^18,19 (Fig. 1).

Figure 1. Schematic representation of a post-transcriptional switch controlling context-specific expression of two alternative gene products.

The mere presence of a miRNA precursor stem–loop in cis may not result in the processing of the miRNA. Adding another layer of complexity, miR-198 belongs to a small cohort of miRNAs that require KSRP for processing. KSRP binds to the GUG motif in the terminal loop of - miR-198 stem-loop precursor within the 3′-UTR of FSTL1 transcript and facilitates processing of miR-198. In the absence of KSRP, the transcript functions as an mRNA making the protein FSTL1, highlighting the essential role of KSRP in switching-on miR-198 processing.¹⁸

However, upon injury, TGF-β signaling turns-off miR-198 processing by down-regulating KSRP and promotes FSTL1 expression. TGF-β down-regulates KSRP through another miRNA, miR-181a. In summary, this complex network regulated by TGF-β and KSRP controls context-specific expression of two alternative gene products from a single transcript that functions either as an mRNA or as a pri-miRNA transcript¹⁸ (Fig.. 1).

Post-transcriptional Control and its Implications in Physiology and Pathophysiology of Diseases

It has been demonstrated that perturbations in RBPs may consequently lead to profound phenotypic alterations in homeostasis resulting in a pathological condition. RBPs and miRNAs co-operatively regulate multiple disease pathways, indicating complementarity between the two types of post-transcriptional regulation. Hereditary hyperferritinemia-cataract syndrome (HHCS) is characterized by bilateral cataracts and increased serum L-ferritin, in the absence of iron overload. Regulation of iron metabolism occurs mainly at the post-transcriptional level. Trans-acting iron regulatory proteins bind to the cis-acting iron-responsive elements, located in the UTRs of target mRNAs and control transcript levels.²⁰ Similarly, regulation of neurofilament protein expression depends on posttranscriptional control of its mRNA including localization, translational efficiency and stability. Imbalances in the expression may lead to formation of neurofilament aggregates resulting in neurodegenerative diseases.²¹ Cyclo-oxygenase-2 (COX-2), aberrantly expressed in multiple stages of colon carcinogenesis, is involved in the synthesis of prostaglandins. In normal intestinal epithelium, this message is degraded by the action of miR-16. However, in colorectal cancers, the mRNA stability factor HuR inhibits miR-16 targeting of COX-2.²² Similarly, in prostate cancer, HuR opposes the repression of ERBB-2 gene expression by miR-331–3p.²³

Adding to this list, we demonstrate a non-functional miR-198/FSTL1 post-transcriptional switch in chronic diabetic ulcer wounds from patients with diabetes mellitus, where wound healing is defective. We found persistent high levels of miR-198 at the chronic wound edge. Furthermore, absence of FSTL1 protein at the wound edge indicates a defective switch in chronic wounds.¹⁸ In conclusion, a non-functional switch leads to impaired keratinocyte migration and loss of re-epithelialization culminating in non-healing chronic wounds, a consequence of defective TGF-β signaling. In patients with diabetes mellitus, chronic wounds are a major health burden resulting in lower extremity amputations²⁴ and modulation of the defective switch may lead to wound re-epithelialization and effective wound healing, resulting in improved patient outcome.

Conclusions

Extensive work on post-transcriptional control of eukaryotic gene expression indicates that mRNA translation is influenced by an array of regulatory mechanisms. The impact of post-transcriptional switches in the dynamic regulation of proteome is increasingly appreciated. Cell-type specific post-transcriptional switches modulate gene expression by multiple modes. These regulatory switches are under the stringent control of growth factors, RBPs and miRNAs. It is challenging to predict the mechanisms involved in the processing of exonic miRNAs. If the mRNA harbors the precursor miRNA stem-loop in its UTR, whether the transcript encodes a functional protein or gets processed into a miRNA is still an open question for multiple exonic miRNAs. Additionally, it would be interesting to see if such a transcript can coordinately express both the encoded protein and the linked miRNA. Complicating the scenario, such post-transcriptional switches are regulated by an intricate network of RBPs and growth factors with extensive cross-talk. Unraveling these networks and elucidating novel post-transcriptional switches may provide an answer for the aberrant gene expression programs in multiple pathological conditions.

Acknowledgments

This work was supported by an A*STAR Investigatorship award to PS, Biomedical Research Council of Singapore and the Skin Biology Cluster Platform, A*STAR.

Submitted

07/14/13

Revised

09/03/13

Accepted

09/05/13

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.