Summary

RNA synthesis, processing and post-transcriptional regulation

Messenger RNA synthesis is accompanied by co-transcriptional addition of two major stability elements. These include 7-methyl guanosine (m⁷G) capping at the 5’ end of the mRNA transcript, which prevents 5’ exonuclease digestion and addition of a variable length poly-A tail to the 3’ end of the mRNA. Splicing of intron-containing transcripts occurs in the nucleus and is mediated by spliceosomal apparati composed of distinctive small nuclear ribonucleoprotein complexes (snRNPs) (1) and amplifies the number of translation products generated from a single genomically templated transcript. Enhancers and repressors of splicing exert additional combinatorial control over other post-transcriptional modifications including RNA editing (1, 2). Following or simultaneous with 5’ and 3’ end modifications and splicing of the nuclear transcript, mRNAs are bound to a complex of nuclear export factors (NXFs) to form a ribonucleoprotein complex (mRNP). The mRNP complex interacts with the nuclear pore complex (NPC) for translocation to the cytoplasm (3). The m⁷G cap is bound by elongation initiation factor 4E (eIF4E) and other cytoplasmic eIF4 proteins, which then engage via eIF3 directly to ribosomes to facilitate mRNA translational initiation (4). Similarly, poly-A binding protein (PABP) also accelerates mRNA translation via activation of the helicase activity of the eIF4 complex (5).

Mechanisms and pathways for RNA degradation

Eukaryotic mRNA half-lives range from minutes to months, the predominant mechanism limiting mRNA longevity being transcript deadenylation. mRNA transcripts earmarked for destruction undergo initial removal of the 5’ m⁷G cap by “decapping” enzymes (6) or alternatively the 3’ poly-A tail may be targeted by deadenylases (7). Poly-A tails are deadenylated at differing rates, mRNAs containing long poly-A tails (> 55 adenosines) being generally more efficiently translated (8). During cellular stress (such as glucose deprivation) (9, 10), or when the mRNA decay machinery is overloaded, mRNA transcripts may be shuttled to cytoplasmic processing bodies (P-bodies) where they are sequestered for degradation or later translation, providing an additional layer of regulation of gene expression and a defense against translating aberrant proteins (11).

In addition to the 5’ cap and 3’ poly-A tail, key mRNA stability elements are found within the 3’ untranslated region (UTR). The 3’ UTR of transcripts manifesting rapid turnover or tight regulation commonly contain AU-rich elements (AREs). Most AREs contain the pentanucleotide AUUUA consensus sequence and are classified as class I or II, reflecting the number and context of pentanucleotides, although non-AUUUA AU-rich elements may also direct mRNA decay (12). ARE domains are targeted by ARE-binding proteins including Hu antigen R (HuR, ELAVL1), tristetraprolin (TTP, ZFP36), apolipoprotein B mRNA editing enzyme, catalytic polypolypeptide 1 (APOBEC1) that modulate mRNA stability (13, 14). CUG triplet repeat, RNA binding protein 1 (CUGBP1) is an ARE-binding protein that modulates mRNA splicing as well as stability and directly recruits the deadenylase poly(A)-specific ribonuclease (PARN), linking AU-rich elements and deadenylation (15). ARE-binding protein mediated mRNA stabilization is incompletely understood, but likely involves competition for binding sites with destabilizing ARE-binding proteins or stabilizing interactions between the poly-A tail and PABP (11). Modulation of ARE-binding protein activity occurs through combinatorial interactions with other signaling pathways including p38 mitogen activated protein kinase (MAPK) and Wnt/β-catenin (16). For example, activation of p38 MAP kinase results in phosphorylation of the ARE-binding protein KH-type splicing regulatory protein (KSRP) in developing myocytes, decreasing the affinity of KSRP for ARE containing mRNAs and thus favoring transcript stabilization (17).

Stability elements within mRNA transcripts may also include microRNA (miRNA)-binding sites (Figure 1A). MicroRNAs are small single stranded non-coding RNAs that are processed and incorporated into the RNA-induced silencing complex (RISC). MicroRNAs are transcribed as mono or polycistronic transcripts to form pri-microRNAs, which are cleaved in the nucleus by the RNAse III endonuclease Drosha to 60–90 nucleotide pre-microRNAs and exported from the nucleus by the karyopherin Exportin 5. Cytoplasmic endonucloeolytic processing by Dicer yields 20–22 nucleotide double-stranded microRNA fragments that bind complementary target mRNAs leading variably (discussed below) to translational suppression, trafficking to P-bodies or degradation (18).

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Figure 1

Mechanisms for therapeutic mRNA targeting

(A) RNA Interference (RNAi). Cellular RNAi machinery may be coopted by a variety of mRNA targeting strategies. Genomically templated pri-miRNA is transcribed in an RNA Polymerase-II (Pol II) dependent fashion and cleaved in the nucleus to pre-miRNA and transported via Exportin 5 to the cytoplasm. Exportin 5 shuttles back to the nucleus, while the pre-miRNA undergoes further processing in the cytoplasm by Dicer. The guide strand is incorporated into the RNA-induced silencing complex (RISC) that includes proteins such as Argonaute (AGO) involved in targeting mRNA and leading to passenger strand degradation. Lentiviral or plasmid encoded short hairpin RNA (shRNA) transcripts may be generated in the nucleus using either Pol II or Pol III driven by a tissue specific promoter/enhancer (TSPE) and are similarly processed to activate RNAi. Exogenously delivered cytoplasmic dsRNA or siRNA may be processed by Dicer and incorporated into the RISC complex and generally require less processing than miRNA or shRNA. Perfect or near perfect complementarity between the guide sequence and target mRNA typically leads to target cleavage. Imperfect complementarity typically results in trafficking of the mRNA to cytoplasmic P-bodies where it is degraded or sequestered for later translation. (B). Antisense oligodeoxynucleotides (ASOs). ASOs may base pair with the target mRNA transcript at almost any location, including the 3’ untranslated region (UTR) which is shown but may also be used to target the 5’ region of the target gene and the initiation AUG codon. Depending on the target location and chemical modification of the ASO, the target mRNA may be cleaved in an RNase H dependent fashion. Alternatively, the ASO may result in steric hindrance of RNA-binding proteins, preventing translation, modifying splicing, or altering mRNA stability. (C) Ribozyme mediated trans-splicing. The guide sequence of the targeting transcript base pairs with the target mRNA upstream of the non-functional or pathologic sequence or mutational site (*). The ribozyme domain of the targeting construct catalyzes splicing of the target transcript to the replacement exons, generating a functional, non-pathogenic mRNA sequence.

Imperfect complementarity between the miRNA and target sequence may result in translational repression. This may occur via alterations in m⁷G cap-dependent recruitment of the 80S ribosome to mRNA (“cap-dependent repression”) and interference with post-initiation translation (“cap-independent repression”) (reviewed in (19)) or alternatively, targeted transcripts may be trafficked to P-bodies for sequestration (20). In contrast, perfect or near-perfect complementarity generally results in transcript destabilization, which may occur either by direct endonucleolytic cleavage within the target sequence (21) or by accelerated deadenylation, 5’ decapping and 5’ exonuclease activity (19). There is still uncertainty however regarding the fate of the target transcript and the role of target-miR complementarity (reviewed in (22)). For example, near-perfect miRNA-target mRNA interactions may lead to translational repression and not cleavage (23). miRNA-mediated translational repression is also reversible as evidenced by the cationic amino acid transporter-1 (CAT-1) which is repressed in human hepatoma cells by miR-122, and reversed by stress (amino acid deprivation or induction of the unfolded protein response). This response is mediated by competition with HuR for binding sites within the 3’-UTR shared with miR-122. These results suggest that miRNA binding and ARE-binding proteins may interact to modulate overall mRNA stability (24).

Mechanisms of Therapeutic Post-Transcriptional RNA Regulation

Hepatotropism of RNA targeting

Early work with modified encapsulated ASOs demonstrated effective hepatotropism after intravenous administration (46) with >80% detectable in the liver at one hour and 26% by four hours. Further experiments demonstrated that subcutaneously injected ASO targeting Fas accumulated in the liver of mice for approximately 10 days (47). Naked siRNAs injected systemically concentrate predominantly in the kidney (48), but modifications to the siRNA backbone increase plasma stability and improve hepatic delivery. Synthetic duplex siRNAs containing one phosphodiester strand and one phosphothioate strand were stable up to 72 hours in 50% mouse serum compared with unmodified single stranded RNA which was degraded in seconds (49). siRNA-lipid complexes including cholesterol, cationic lipid or lipidoid nanoparticles increase transfection efficiency (50) and have been used to optimize in-vivo targeting of apolipoprotein B (APOB) and proprotein convertase subtilisin/kexin type 9 (PCSK9) (discussed below) (51–53).

Potential for off-target effects and toxicity

siRNAs activate the immune system (54) and double stranded RNA can activate the intracellular receptors dsRNA-dependent protein kinase R (PKR, EIF2AK2) or retinoic acid inducible gene-I (RIG-I, DDX58). Activation of PKR results in global suppression of protein synthesis, ultimately leading to apoptosis. Activation of either PKR or RIG-I leads to interferon production and other antiviral responses, including Toll-like receptors TLR7 and TLR8 which are activated by siRNA in a sequence and delivery-specific fashion. U- and G- rich siRNAs, longer dsRNA sequences, single stranded siRNAs as well as cationic lipid complexed siRNAs result in the most potent activation of immunostimulatory signaling pathways. Conversely, shorter siRNA sequences, low G/U content, double stranded, naked and cholesterol-complexed siRNAs appear to be least immunostimulatory (54). In-vivo trials of siRNA therapy have utilized chemical modifications such as 2 nucleotide 3’ overhangs and 2’-O-methyl modifications that diminish immunostimulatory effects of siRNAs as evidenced by attenuated production of interferon-α (IFNA2), interleukin-6 (IL6), and tumor necrosis factor-α (TNF) (51, 55).

RNA therapy mediated off-target effects can also occur via sequence homology to unintended transcripts. ASOs targeting protein kinase C-α RNA suppressed protein kinase C-ζ (PKC-ζ, PRKCZ) mRNA even though these isoforms share only 11/20 base pair homology (56). Additionally, while siRNA binding to target mRNA with perfect complementarity results in mRNA cleavage, imperfect complementarity suppresses translation, similar to that discussed above with miRNA (57).

Experience with RNAi therapy using adeno-associated virus (AAV) has revealed potential hepatotoxicity. AAV mediated shRNA expression targeting transgenically expressed human α-1 antitrypsin (AAT) mRNA (43) suppressed h-AAT mRNA, but was complicated by lethal hepatotoxicity, likely due to oversaturation of Exportin-5 as alluded to above (Figure 1A). Accordingly, successful delivery of an RNAi effector must be accompanied by careful modulation of intracellular concentrations in order to avoid toxicity.

Therapeutic Applications of RNA Modulation in Metabolic Liver Disease

Efforts are underway to utilize RNA-targeting therapy in metabolic conditions involving the liver, in particular the management of hypercholesterolemia, where despite the success of statin-based therapies, only 2/3 of patients achieve their target low density lipoprotein (LDL) goal (58). Patients with hereditary mutations in either the LDL receptor (familial hypercholesterolemia, FH) or in the LDL receptor binding region of ApoB100 fail to clear ApoB100-containing lipoproteins from the serum, leading to accumulation of lipid particles and hypercholesterolemia. ASO targeting of ApoB-100 has been used in proof-of-principle experiments in ApoE-deficient and LDLR-deficient mice, (59), where cholesterol reduction ranged from 25–55%, with LDL-C reductions as high as 88% without hepatic steatosis. A double blind, placebo controlled, randomized trial of mipomersen (ApoB ASOs) in 36 volunteers with mild sporadic dyslipidemia demonstrated decreased serum LDL and ApoB levels of 35% and 50% respectively. There were no serious adverse events with the most common side effect being mild injection site erythema (60). More recently, phase 3 studies with mipomersen in FH subjects revealed a 25% reduction of LDL cholesterol levels after 26 weeks of treatment (32, 61). Further studies are directed toward assessing whether this approach places patients at risk for hepatic steatosis as seen in patients with some naturally occurring ApoB mutations or with familial hypobetalipoproteinemia (62).

Targeting the 3’ end of ApoB mRNA with cholesterol-conjugated siRNA in mice lowered serum ApoB levels and decreased serum LDL (52) and stable nucleic acid lipid particles (SNALP)-formulated siRNA targeting the 5’ end of ApoB mRNA (53) in cynomolgus monkeys suppressed ApoB mRNA by 90% at 48 hours after a single dose, an effect that persisted for 11 days, accompanied by >60% reductions in serum cholesterol.

ASO-mediated therapeutic ApoB RNA splicing inferference was used to induce skipping of exon 27 (38) to generate a truncated ApoB isoform predicted to decrease LDL-cholesterol. The “skip 27” mRNA translated product (ApoB87) was exported from the cells at decreased efficiency compared to ApoB100, an effect reminiscent of that associated with familial hypobetalipoproteinemia (FHBL) patients expressing an APOB truncating mutation (63). Differential processing of the truncated ApoB mRNA and alterated posttranslational lipidation of the protein product might account for the therapeutic efficacy of this targeting strategy.

A second target of hypolipidemic therapy is proprotein convertase subtilisin/kexin type 9 (PCSK9), which regulates hepatic LDL-receptor expression via altered protein processing (64). Gain-of-function mutations result in increased degradation of the LDL-receptor and increased lipid levels. Conversely, loss-of-function mutations result in increased expression of LDL-receptors and decreased serum cholesterol (65). Epidemiologic studies link mutations in PCSK9 to low LDL cholesterol levels in approximately 2% of African Americans (66), with an approximately 40% reduction in LDL-cholesterol and reduction in coronary artery disease (64), making PCSK9 an attractive hypolipidemic target. In-vivo targeting of PCSK9 using ASOs in hyperlipidemic mice fed a high fat diet increased hepatic LDL receptor expression and decreased serum cholesterol (67). siRNA targeting of PCSK9 in rodents and non-human primates using intravenous lipidoid nanoparticles also decreased serum cholesterol (51).

ASOs have also been developed (antagomirs) to target miRNAs involved in hepatic lipid metabolism, including miR-122. In-vivo silencing of miR-122 in mice using a miR-122 antagomir, decreased 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMG-CoA reductase, HMGCR) and acyl-Co A synthetase 2 (ACSS1) among other targets, accompanied by a >40% decrease in plasma cholesterol and without adverse effects on hepatic function or elevated transaminase levels (45). Analysis of miRNA expression patterns in patients with non-alcoholic steatohepatitis (NASH) revealed reduced expression of miR-122 with corresponding alterations in several target genes that were replicated in HepG2 cells following either miR-122 knockdown or overexpression (68). These findings and the implications for NASH pathogenesis need to be interpreted with caution however, since miR-122 is known to modulate the expression of ~200 target genes (69) and the precise mechanisms by which alterations in miR-122 expression influence hepatic lipid metabolism in human subjects remains to be defined.

Immune-Mediated Liver Disease

Hepatocytes are susceptible to Fas-mediated apoptosis and disruption of this pathway protects hepatocytes from acute liver injury. Mice treated with ASOs targeting Fas (FAS) were protected from Fas antibody-induced fulminant liver failure and from a sub-lethal dose of acetaminophen (47). Similar experiments using RNAi confirmed that interference with Fas signaling protects from fulminant hepatitis (70). Taken together, these data strongly suggest that RNA-directed therapy may have clinical utility in treating disorders of immune-mediated liver injury as well as fulminant liver failure with an immune-mediated component.

Future Directions and Prospects

The last several years have witnessed great advances in understanding of the physiologic and pathologic roles of regulated mRNA stability. Among the challenges moving forward will be to define how aberrant post-transcritional mRNA processing including mRNA trafficking and stability functions in disease pathogenesis and to define optimal mRNA targets for specific clinical conditions. From the standpoint of drug development, refining potency, specificity, minimization of side effects and tissue delivery will be crucial goals in coming years.

Acknowledgements

REFERENCES