In an analysis of just five of the hundreds of RBPs encoded by the S. cerevisiae genome, we found that more than 700 transcripts appeared to be specifically bound by one or more RBPs, with each of the five Puf family proteins “tagging” a distinct set of mRNAs. These sets encode functionally and cytotopically related proteins. For three of the Puf proteins, we identified distinct short sequences in the associated specific set of mRNAs, typically in the 3′-UTR, which were sufficient for specific binding to the cognate Puf protein in vivo. Many sets of mRNAs encoding proteins localized to the same subcellular compartment, protein complex, or functional system were bound by the same Puf protein. Puf3p, which specifically associated with cytoplasmic mRNAs encoding mitochondrial proteins, generally affected the steady-state levels of its mRNA targets as reflected by their increased abundance in puf3 mutant cells.
The selective “tagging” by sequence-specific RBPs of mRNAs that share common physiological roles suggests a general and widespread mechanism for coordinated control of their expression. Previous reports have identified coordinated regulation of small sets of functionally related mRNAs by specific RBPs. For example, mammalian stem–loop binding protein (SLBP) associates with all five classes of histone mRNAs and guides proper 3′-end formation (Dominski and Marzluff 1999
). Iron regulatory proteins (IRPs) bind to and regulate translation of five different mRNAs encoding proteins involved in iron metabolism (Eisenstein and Ross 2003
), and a cytoplasmic poly(A) polymerase regulates multiple mRNAs in early development (Mendez and Richter 2001
). Based on these and other examples (Tenenbaum et al. 2000
), Keene and Tenenbaum (2002
) have suggested that messenger RBPs could define “post-transcriptional operons.” Our results provide strong support for this general idea of coordination of gene expression via RBPs and suggest that the post-transcriptional control afforded by combinatorial binding of RBPs to mRNAs could allow greater regulatory flexibility than a simple operon (see also Keene and Tenenbaum 2002
). Further, we suggest that RBPs may play important roles in subcellular localization and efficient assembly of protein complexes.
The RBPs encoded in eukaryotic genomes rival specific transcription factors in their numbers and diversity, raising the intriguing possibility that specific regulation of the localization, translation, and survival of mRNAs might be comparable in their richness and complexity to regulation of transcription itself. Each of the five Puf proteins interacts with a distinct large set of mRNAs, comprising more than 700 different mRNAs in total. Five other RBPs in S. cerevisiae
have been subjected to a similar genome-wide survey of their mRNA targets. She2p, which plays a critical role in selective targeting of specific mRNAs to the bud tip (Shepard et al. 2003
), Khd1p, which has also been implicated in localizing gene expression to the nascent bud (A. P. Gerber, unpublished data), and Scp160p, an RBP implicated in genome stability (Li et al. 2003
), were each found to bind from 20 to hundreds of distinct mRNAs, and two proteins implicated in RNA export from the nucleus, Yra1p and Mex67p, were each associated with more than 1,000 mRNAs (Hieronymus and Silver 2003
). Thus, just ten of the 567 S. cerevisiae
proteins known or predicted from the genome sequence to have RNA binding activity (Costanzo et al. 2001
) have been found to bind, in a functionally specific pattern, a total of approximately 2,500 different transcripts (approximately 40% of the transcriptome). The extent and specificity of the RNA–protein interactions represented by the proteins studied to date, extrapolated to the hundreds of putative RBPs that remain to be investigated, suggest the existence of an extensive network of RNA–protein interactions that coordinate the post-transcriptional fate of large sets of cytotopically and functionally related RNAs through each stage of its “lifecycle.” It further suggests a potential regulatory repertoire comparable in its diversity and richness to that of the DNA-binding transcription factors (). Indeed, the combinatorial binding of mRNAs by multiple RBPs could, in principle, define a specific post-transcriptional fate for each individual mRNA (for an example, see Sonoda and Wharton 2001
Specific Proteins Bind Functional Groups of Genes for Regulation
Many sets of mRNAs bound by the same Puf protein encode proteins that act in the same subcellular location, form stochiometric complexes, or are implicated in the same cellular pathway. This organization is most clearly exemplified by Puf3p, which selectively bound mRNAs encoding mitochondrial proteins, including at least 70% of all mitochondrial ribosomal proteins (see ). Combinations of RBPs could specify smaller sets of RNAs encoding more precisely defined functional groups of proteins. For example, the mRNAs encoding the core histone proteins were among the small set of mRNAs that were associated with both Puf4p and Puf5p. These results therefore hint that networks of functional and physical interactions among proteins could be reflected in a corresponding network of mRNA–protein interactions that coordinate post-transcriptional control of their expression and fate.
For three of the Puf proteins, we found that RNA–protein interactions were directed by compact sequence elements, usually located in the 3′-UTR of the mRNA (see ). Interactions with 3′-UTR sequences have been described for many cytoplasmic RBPs involved in post-transcriptional regulation (Mazumder et al. 2003
). Our analysis has revealed that such recognition elements are probably much more widespread than previously recognized. Sequence and structural elements in mRNAs that are related to the function or cellular localization of the encoded proteins may be a general feature of eukaryotic genes, paralleling the role of the DNA sequences that direct specific transcription factors to promoters and enhancers (Cliften et al. 2003
The multifocal cytoplasmic distribution of Puf proteins raises the possibility that the mRNAs associated with each Puf protein are colocalized (see ). In mammalian cells, specific mRNA molecules and specific messenger RBPs have also been found to be localized to specific “granular” subcytoplasmic loci, although the generality of this phenomenon has not been established (Andersen and Kedersha 2002
; Eystathioy et al. 2002
; Farina et al. 2003
). One function of the Puf proteins and related proteins that bind specific families of mRNAs could be to localize functionally related mRNAs to specific cytoplasmic loci. Physical clustering of functionally related groups of mRNAs could aid the assembly of complexes and the coordinated control of translation or RNA turnover. In support of this idea, it has recently been suggested that mRNA decay in the cytoplasm of S. cerevisiae
occurs in distinct loci (Sheth and Parker 2003
) and, further, that mRNAs encoding different subunits of stoichiometric complexes do indeed have concordant decay rates (Wang et al. 2002b
). We propose that the location in the cell at which any mRNA is translated or degraded is not left to chance. Instead, every mRNA that leaves the nucleus may be delivered, in a process directed by specific protein–RNA interactions, to one of a limited number of specific foci in the cytoplasm, designated as destinations for a specific functionally related family of mRNAs. These foci could serve to colocalize and coregulate synthesis of proteins that need to assemble or act together, thereby facilitating efficient and rapid assembly and localization of the proteins. The number of distinct families of functionally specialized foci may be quite large. The locations of these foci need not correspond to recognizable cellular features, but may simply be ad hoc sites for localized, coordinated translation of proteins that are to be assembled into a complex or a functional unit. Specific predictions of this hypothesis, such as colocalized translation of the subunits of stoichiometric complexes, should be amenable to direct experimental tests.
Combinatorial binding of mRNAs by specific regulatory proteins, linking their post-transcriptional regulation to specific signal transduction pathways, could allow rapid and efficient reprogramming of gene expression during development or in response to changing physiological conditions. Indeed, regulation of specific genes by external signals via RPBs has been described in higher eukaryotes (Lasko 2003
). For example, the signal transduction and activation of RNA (STAR) proteins contain RNA-binding motifs combined with protein–protein interaction domains and phosphorylation sites, which could allow integration of stimuli conducted by signal transduction cascades (Lasko 2003
). Similarly, the Puf proteins contain numerous putative phosphorylation motifs, as well as domains with characteristics often implicated in protein–protein interactions, such as glutamine/arginine-rich regions (Michelitsch and Weissman 2000
) (see ).
Coordination of cellular processes has long been thought to be mediated primarily at the transcriptional and post-translational level. Our results join a growing body of studies (Tenenbaum et al. 2000
; Eystathioy et al. 2002
; Wang et al. 2002b
; Hieronymus and Silver 2003
; Shepard et al. 2003
; see also Keene and Tenenbaum 2002
) that suggest that the localization, translation, and stability of mRNAs are subject to extensive and important regulation and coordination by interaction with a diverse set of RBPs. Systematic mapping of these interactions and deciphering their roles, molecular mechanisms, and coordination will undoubtedly yield important new insights into biological regulation and the gene expression program.