RACK1 and the microRNA pathway

Abstract

MicroRNAs (miRNAs) control many aspects of development and adaption in plants and in animals by post-transcriptional control of mRNA stability and translatability. Over the last years numerous proteins have been identified in the miRNA pathway. The versatile scaffold protein RACK1 has been associated with efficient miRNA production and function in plants and metazoans. Here, we briefly summarize the differences of RACK1 function in the plant and animal miRNA pathways and discuss putative mechanisms and functional roles of RACK1 in miRNA biogenesis and action.

MiRNA biogenesis in animals and plant

Although the plant and animal miRNA pathways differ in respect to the processing location, the involvement of auxiliary proteins, and the main mode of action of the mature miRNA, some key steps are strikingly similar.^1,2 In animals and plants, mature miRNA duplexes are released from longer primary-microRNA (pri-miRNA) transcripts with hairpin-like structures by the activity of RNaseIII-like proteins. Animal pri-miRNAs are first processed in the nucleus into precursor-miRNAs (pre-miRNAs) by DROSHA and an auxiliary protein, PASHA/ DIGEORGE SYNDROME CRITICAL REGION 8 (DGCR8).^3,4 Pre-miRNAs are then transported into the cytoplasm and further processed by DICER and partners (e.g., TRANS-ACTIVATION RESPONSE RNA BINDING PROTEIN (TRBP) to release mature miRNA duplexes.^4,5 In plants, processing of pri-miRNAs into mature miRNA duplexes is performed exclusively in the nucleus by DICER-LIKE 1 (DCL1) which is assisted by other RNA-binding proteins like SERRATE (SE) or HYPONASTIC LEAVES 1 (HYL1).^6-12 Animal and plant mature miRNA duplexes are loaded into ARGONAUTE (AGO) effector complexes facilitated by the chaperones HEAT-SHOCK-PROTEIN 90 and 70 (HSP90 and HSP70).^13-16 The AGO complex guided by the miRNAs binds and regulates target mRNAs.^1,2

The scaffold protein RACK1

In the beginning of the 1990s RECEPTOR FOR ACTIVATED C KINASE (RACK1) has been discovered as an interactor of an active form of PROTEIN KINASE C (PKC).¹⁷ RACK1 is present exclusively in eukaryotes and structural analyses of yeast, human and Arabidopsis RACK1 revealed that RACK1 proteins form a 7 bladed β-propeller structure with each blade containing one WD40 repeat.^18-20 RACK1 proteins from different species share 43–73% identical amino acids, demonstrating the high conservation of RACK1 across kingdoms.²¹ RACK1 scaffold proteins have no catalytic activity; instead, they bridge interactions between proteins, they influence the stability and activity of interactors or their cellular localization.²¹ A vast diversity of RACK1 binding partners have been reported in yeast, animals and plants ranging from, kinases, phosphatases, channels, receptors, transcription factors to the eukaryotic 40s ribosomal subunit.^21-24 Due to its plethora of interaction partners, RACK1 is believed to act as signal integrator, which interconnects distinct signaling pathways to control essential cellular processes such as transcription and translation, cell proliferation and growth as well as cell spreading and cell-cell interactions.^21-23

Animal and plant RACK1 proteins affect the miRNA pathway

Three reports described the functions of RACK1 scaffold proteins in the miRNA pathway of C. elegans, humans, and Arabidopsis.^25-27 Although RACK1 is an important factor in the miRNA pathway, the exact functions of RACK1 seem to vary in all organisms (Fig. 1, Table 1).

Figure 1. Model for RACK1 function in the plant and metazoan miRNA pathway. (A) RACK1 influences different steps of miRNA biogenesis and action. Green = plant proteins, Blue = Metazoan proteins, Grey = Common factors. (B) Exogenous or endogenous signals may affect the miRNA pathway by either modulating RACK1 abundance or function. This may lead to changes in miRNA expression and function in response to different stimuli.

Table 1. Summary of RACK1 functions in the miRNA pathway.

	C. elegans	human	Arabidopsis
Early miRNA biogenesis	-	-	T
AGO complex maturation	-	T	?
AGO function	T	-	?
RACK1 interactions with miRNA components	AGL1	KSRP AGO2	SE AGO1
References	27	26	25

T, proven function; ?, possible function, -: unknown

In Arabidopsis, we found RACK1 to be interacting with the miRNA processing component SERRATE, suggesting a role for plant RACK1 in miRNA processing²⁵ (Fig. 1A). Indeed, molecular analyses of rack1 mutant plants show that miRNA accumulation is globally decreased like in other well-studied miRNA mutants.^7,25,28,29 In line with this observation, miRNA-mediated repression of target mRNAs is released in rack1 mutants. In addition, RACK1 is found in complexes with the Arabidopsis AGO protein involved in miRNA action, AGO1, which provides a hint that plant RACK1 may also function during later steps of the plant miRNA pathway (Fig. 1A).

Also the processing of metazoan pri-miRNAs is aided by auxiliary protein, among them KH-TYPE SPLICING REGULATORY PROTEIN (KSRP), which has been shown to interact with RACK1 in humans (Fig. 1A). KSRP a single-strand nucleic acid binding protein, which binds to the terminal loop of a subset of animal pre-miRNAs and promotes processing of the bound pre-miRNA by interacting with the nuclear DROSHA and the cytoplasmic DICER complexes.³⁰ However, the amounts of KSRP dependent mature miRNAs remain unchanged in rack1 knock-down cells, indicating RACK1 is not involved in processing of KSRP dependent miRNAs. Because artificially delivered mature miRNA are fully functional in rack1 knock down cell lines, the authors hypothesized RACK1 promotes the transfer and recruitment of miRNAs from processing complexes into AGO complexes.²⁶

In a third publication, the authors identified RACK1 as a direct binding partner of the C.elegans AGO, AGL-1.²⁷ RACK1 is important for miRNA-mediated silencing of target mRNAs probably by recruitment of AGO complexes to the translating mRNA by ribosomal RACK1²⁷ (Fig. 1A). In agreement with this hypothesis, AGO occupancy to polysomes is reduced in rack1 deficient cell lines.²⁷

In summary, animal and plant RACK1 seem to influence the miRNA pathway via several distinct mechanisms, including early steps of the miRNA biogenesis as well as AGO dependent processes. Puzzling out RACK1’s molecular mode of action in the miRNA pathway will be an important issue of future research.

Possible mechanisms and functions of RACK1 in the miRNA pathway

Protein-protein interactions play key roles in the progression and the regulation of the plant and animal miRNA pathway. As RACK1 acts as scaffold protein, it could function as a molecular glue to tighten interactions between key miRNA factors or to direct regulatory factors to the miRNA pathway. Posttranslational modifications of miRNA factors are key regulatory events in animal and plant miRNA production and function. Phosphorylation of the RNA-binding proteins HYL1 and TRBP, co-factors of DCL1 and DICER, respectively, has been shown to influence miRNA biogenesis in plants and animals.^31,32 In humans, the nuclear localization of DROSHA is dependent on its phosphorylation status and also a phosphorylated peptide of the plant DCL1 protein has been discovered.^33,34 A variety of post-translational modifications have been reported for animal and plant AGO proteins: hydroxylation and ubiquitination influence AGO stability, polyADP-ribosylalation inhibits miRNA-mediated target regulation upon stress and phosphorylation affects AGO localization and miRNA binding.^35-37 As RACK1 binds a variety of phosphatases, kinases and has a role in ubiquitination,^21,38 RACK1 scaffold proteins might transiently recruit regulatory factors to ensure efficient miRNA production and function.

A possible biological role of RACK1 in the miRNA pathway comes from the analogy of RACK1’s function as part of the 40S ribosomal subunit. There, RACK1 is thought to act as a signal integrator that transfers environmental cues such as stress to the translating ribosome.²² Also the production, the stability and function of mature miRNAs is influenced in a spatial, time, and stimuli dependent manner.^8,31,39-42 Several studies revealed that RACK1 expression is dynamically changed upon different exogenous and endogenous signals.^21,26,43-46 Also posttranscriptional modifications, including phosphorylations, have been reported to influence RACK1`s ability to modulate the function of interacting proteins.²¹ Hence, changes in RACK1 levels or post-translational modifications could contribute to shape miRNA expression patterns in response to environmental cues (Fig. 1B).