Defining potentially conserved RNA regulons of homologous zinc-finger RNA-binding proteins

Abstract

BackgroundGlucose inhibition of gluconeogenic growth suppressor 2 protein (Gis2p) and zinc-finger protein 9 (ZNF9) are conserved yeast and human zinc-finger proteins. The function of yeast Gis2p is unknown, but human ZNF9 has been reported to bind nucleic acids, and mutations in the ZNF9 gene cause the neuromuscular disease myotonic dystrophy type 2. To explore the impact of these proteins on RNA regulation, we undertook a systematic analysis of the RNA targets and of the global implications for gene expression.ResultsHundreds of mRNAs were associated with Gis2p, mainly coding for RNA processing factors, chromatin modifiers and GTPases. Target mRNAs contained stretches of G(A/U)(A/U) trinucleotide repeats located in coding sequences, which are sufficient for binding to both Gis2p and ZNF9, thus implying strong structural conservation. Predicted ZNF9 targets belong to the same functional categories as seen in yeast, indicating functional conservation, which is further supported by complementation of the large cell-size phenotype of gis2 mutants with ZNF9. We further applied a matched-sample proteome-transcriptome analysis suggesting that Gis2p differentially coordinates expression of RNA regulons, primarily by reducing mRNA and protein levels of genes required for ribosome assembly and by selectively up-regulating protein levels of myosins.ConclusionsThis integrated systematic exploration of RNA targets for homologous RNA-binding proteins indicates an unexpectedly high conservation of the RNA-binding properties and of potential targets, thus predicting conserved RNA regulons. We also predict regulation of muscle-specific genes by ZNF9, adding a potential link to the myotonic dystrophy related phenotypes seen in ZNF9 mouse models.

RNA regulons and the RNA-protein interaction network

“…For example, in a large-scale study defi ning the mRNA targets for more than 40 yeast RBPs, the potential cognate consensus recognition sequences could be deduced by bioinformatic methods for 14 RBPs (14,82) . Similar global RIP-Chip approaches led to the identifi cation of many binding sites for RBPs in different species (28,77,83) . A well-characterized example of an evolutionarily conserved sequence recognition mode is provided by the PUF family of RBPs, whose members bind to single-stranded UGUAAAUA or closely related octamer motifs via eight repeats of the PUFhomology domain (67, 84 -86) .…”

“…The protein contains seven retroviral type ZnF RNAbinding domains and binds to G(A/U)(A/U) repeats that are mainly located in the coding regions of hundreds of mRNA targets that can be grouped into functional categories such as rRNA processing, ribosomal function, and cell motility. The matched-sample analysis of the changes of mRNA steadystate levels and protein levels upon Gis2p overexpression revealed distinct outcomes for functionally related subsets of mRNA targets (77) . Thus, the various mRNA targets of Gis2p appear to be grouped into different RNA regulons that are differentially regulated.…”

“…However, to date only a few studies have addressed directly and comprehensively how this extrapolates to changed protein levels. For example, we have recently performed a combined transcriptomic and proteomic analysis to measure the effects of the yeast RBP glucose inhibition of gluconeogenic growth suppressor 2 (Gis2) on experimentally defi ned target mRNAs (77) . The protein contains seven retroviral type ZnF RNAbinding domains and binds to G(A/U)(A/U) repeats that are mainly located in the coding regions of hundreds of mRNA targets that can be grouped into functional categories such as rRNA processing, ribosomal function, and cell motility.…”

“…Likewise, the application of PAR-CLIP to identify the mRNA targets for FUS and EWSR, which are highly conserved member of the FET family of RBPs with important roles in oncogenesis and neuronal diseases, revealed a combination of structural and sequence elements in the RNA target that directs the binding of these proteins to it (58) . The implementation and refi nement of transcriptome-wide analysis and bioinformatics tools [e.g., (90) ] to reveal the recognition motifs of post-transcriptional regulators and to decipher the underlying RNA code, remains a major challenge for the future [e.g., (77,83,91) ].…”

La-motif–dependent mRNA association with Slf1 promotes copper detoxification in yeast

“…RIP of tagged proteins was carried-out as described (Scherrer et al 2011). In brief, 1 L cultures of cells expressing endogenously tagged SLF1-, SRO9-, or FPR1-TAP (negative control) were grown in SC to an OD 600 of 0.6-0.9 at 30°C, while cells overexpressing WT or LAM-m Slf1-6His-HA-ZZ on a plasmid were grown in SCgal-Ura.…”