mRNA-binding proteins and cell cycle progression

Abstract

Proteins that bind to each mRNA may affect the latter’s abundance and location in the cell and how well ribosomes will translate that mRNA into a protein. Hence, mRNA-binding proteins (mRBPs) represent obvious control points in gene expression. Surprisingly, little is known about mRBPs and cell-cycle progression.

Why the roles of mRBPs in cell division are significant

Interactions between mRBPs and a primary transcript could determine how well the transcript is processed to its mature, translatable form, the transcript’s stability and overall abundance, and how well ribosomes will translate the transcript into a protein. In budding yeast – based on extensive profiling of cell cycle-dependent changes in mRNA [1,2] and protein [3] levels – the abundance of 15–20% of mRBPs changes in the cell cycle (Table S1A in the supplemental information online). As I will discuss here, mRBPs are not only regulated in the cell cycle but also control cell-cycle progression.

Especially for mRNAs known to be translated with different efficiencies during cell division [4], mRBPs are expected to be responsible for much of that control for two reasons. First, the overall rate of protein synthesis does not change during the cell cycle in budding yeast [5], and only slightly, if at all, in animal cells [6]. Second, there is no evidence that the concentration of ribosomes or codon usage changes during cell division [3]. In shifting nutrients and environments, translational control mechanisms that depend on global changes in the availability of the protein synthesis machinery can be very effective [7]. However, such mechanisms are unlikely to underpin cell cycle-dependent translational control in unperturbed cycling cells. Hence, mRBPs become the likely, perhaps even necessary, players to impart that control.

mRBPs: what kind and how many?

It is necessary to decide on the mRBP set to be interrogated for roles in the cell cycle. On the one hand, the broadest approach is to include the entire predicted universe of proteins that could bind RNAs. However, that number is substantial, estimated to be about 600 in budding yeast, 10% of all the proteins in that organism [8] (Table S1B). On the other hand, one could limit the analysis to proteins shown to physically interact with mRNAs in the cell [9] (Table S1B). Based mostly on such experimental evidence, there are now 176 proteins listed in the Saccharomyces Genome Database (SGD) as proteins that bind mRNA (Table S1B).

Here, an intermediate approach was used, including all the mRBPs identified experimentally by Mitchell et al. [9] and many of the ones predicted by Hogan et al. [8]. I excluded, however, mRBPs that carry out their mRNA-binding function in settings (e.g., inside mitochondria) that do not apply to the mRNAs in the rest of the cell, ribosomal proteins, or other mRBPs that are highly specialized (e.g., Est1,2p associated with the telomerase RNA). Overall, a set of 308 yeast mRBPs were used in the analysis (Table S1A,B). Of note, 248 of these 308 yeast mRBPs (80%) are conserved in animals (Table S1A).

Expression of mRBPs in the cell cycle

Among the 308 mRBPs, the abundance of 54 of them may change in the cell cycle. Forty-three mRBP transcripts are among the 1000 most periodic transcripts, their levels peaking at distinct phases of the cell cycle (Table S1A: I took their periodicity scores from Santos et al. [2], the lower the score the more periodic the transcript’s abundance). This group of mRBPs is enriched for gene products involved in RNA processing (GO:0006396; 31 of the 43 transcripts, P = 1.6e-17, with Holm-Bonferroni test correction) and noncoding RNA processing (GO:0034470; 27 of 43 transcripts, P = 4.7e–16). In addition to transcriptomic profiling, cell cycle-dependent changes in the proteome of synchronously proliferating yeast cells have been recently reported [3], with the abundance of 13 mRBPs changing in the cell cycle (Table S1A).

The most periodic mRBP transcript is YBL111C, encoding a nonconserved putative helicase-like protein within the telomeric Y’ element. However, it is not clear whether these elements yield functional gene products [1]. The next most periodic mRBP transcript is YLL032C, with a peak expression in the G2 phase. YLL032C codes for a conserved cytoplasmic protein of unknown function, estimated to bind ~80 mRNAs in the cell, and enriched for a putative binding motif with sequence (A/U)AUACC(C/U) [8]. Yll032Cp is a K homology (KH) domain protein, a structural motif known to mediate RNA binding. The human ortholog of YLL032C, HDLBP, encodes a protein that binds RNA and induces heterochromatin formation; it also binds lipoproteins. Interestingly, among the targets of Yll032Cp is MIH1 [8], encoding a protein tyrosine phosphatase that in mitosis dephosphorylates and activates the master regulator of cell division, the cyclin-dependent kinase Cdk (see Glossary).

Overview of cell-cycle phenotypes of mRBP mutants

The case of Cdc40p, a splicing component, reflects the multiple intersections of splicing with cell-cycle progression. The cdc40 temperature-sensitive mutant was identified initially in classic screens of the cell cycle because when shifted to its non-permissive temperature it arrests in the G2 phase as large budded cells. Nevertheless, before they arrest in the G2 phase, cdc40 mutants are also delayed at the G1/S transition, and progress through the S phase more slowly [10]. Overall, for 55 of the 308 mRBPs, their loss-of-function mutants display one or more cell cycle-related phenotypes (including changes in budding and cell size) (Table S1A).

A delay in the G1 phase is seen in 21 mRBP mutants, and another ten have a small cell size (Table S1A). Twelve mRBP loss-of-function mutants display an arrest or delay after the cells complete the G1/S transition (Table S1A). Loss of Rrp12p (involved in the maturation of SSU-rRNA from the rRNA transcript), Rat1p (a 5′-to-3′ RNA exoribonuclease), or Pap2p (a noncanonical poly(A) polymerase), leads to an S phase delay or arrest. Loss of Syf1p, Clf1p, or Cef1p (all essential splicing factors) arrests the cell cycle at the G2/M transition, similarly to the cdc40 mutants mentioned above. In other mRBP mutants, a delay in the G2 phase is evident upon loss of Ebp2p (involved in rRNA processing), while an M phase delay is seen in cells lacking Bfr1p, which brings mRNAs to cytoplasmic P bodies, or mutants lacking Pat1p, a deadenylation factor with multiple roles, including in P-body assembly. A remarkably specific cell-cycle phenotype is observed in cells lacking Sup45p, a translation termination release factor (eRF1) that also localizes to P bodies. SUP45 is essential, and temperature-sensitive mutants arrest in cytokinesis due to a defect unrelated to the function of Sup45p in translation termination [11].

One could ask whether the observed phenotypes reflect general, cell growth-related roles, usually manifest with a delay in the G1 phase, lower budding index, and likely a small cell size. Such outcomes, for example, are seen in the vast majority of loss-of-function mutants of the protein synthesis machinery [12]. In contrast, however, the cell-cycle phenotypes of mRBP mutants are more varied at multiple cell-cycle transitions, suggesting that binding to specific mRNAs encoding gene products with roles in cell division may underpin some of the observed phenotypes. Two mRBP ‘case studies’ well understood for their role in cell division are highlighted in Figure 1 (Whi3p, a yeast mRBP) and Figure 2 (DENR-MCT1, a mammalian heterodimeric mRBP). These two mRBPs are also Cdk targets, and their mRNA-binding partners encode gene products with roles in the cell cycle. But there are likely more such examples. Among the 308 mBRPs analyzed here, 38 may be Cdk substrates (Table S1A, based on available information in SGD and Cyclebase [2]). mRBPs targeted by Cdk provide additional potential ‘handles’ for the cell-cycle machinery to influence cell-cycle progression through mBRPs.

Figure 1. Whi3p inhibits CLN3 expression and delays the G1/S transition.

A conserved budding yeast mRNA binding protein (mRBP), Whi3p – which has periodic expression, cell cycle-associated phenotypes, and is phosphorylated by Cdk – targets the CLN3 mRNA. Cln3p is a G1 cyclin, a well-known and potent regulator of cell size and the G1/S transition. Whi3p binds to 3’-end (U/G)CAU motifs, destabilizes the CLN3 tran script, and binds to 5’-end (U/G)CAU motifs to inhibit translation of CLN3. Despite the many mRNA targets of Whi3p, its interaction with CLN3 mRNA appears to account for most of its mRBP roles in cell division [14].

Figure 2. Mitotic phosphorylation of DENR derepresses translation of target mRNAs in mitosis.

In animals, DENR-MCT1 is a heterodimer protein complex that derepresses the translation of specific mRNAs, which carry inhibitory upstream open reading frames (uORFs). DENR–MCT1 (analogous to Tma22p/Tma20p in yeast) promotes ribosome recycling and reinitiation, enabling the synthesis of the main gene product. It turns out that mitotic Cdk/cyclin complexes (Cdk1/cyclinB and Cdk2/cyclinA) phosphorylate and protect DENR from mitotic degradation [15]. As a result, the translation of mRNAs targeted by DENR–MCT1 is derepressed, ensuring the timely and faithful completion of mitosis. When all Cdk/cyclin activity is turned off as cells exit mitosis, the DENR phosphorylation is reversed, DENR is degraded, and uORFs inhibit translation of the downstream ORF.

Concluding remarks

To answer the question of how mRBPs control cell-cycle progression, it is necessary to identify the mRBPs that bind to a transcript encoding a gene product with cell-cycle roles at different times during cell division, and then to interrogate in detail the functional significance of those interactions. However, profiling the repertoire of mRNAs associated with a given mRBP during the cell cycle, using highly synchronous cells, is largely unstudied. Given that most mRBPs bind many mRNAs (a complexity that only gets amplified in multicellular eukaryotes), it would be a major challenge to identify those mRNAs most relevant for cell-cycle progression. Furthermore, even from asynchronous cells, it is much easier to ask and answer what mRNAs a specific mRBP might bind to rather than the other way around. Until recently, the technology for ‘mRNA-centric’ physical association studies did not exist. Now, however, Cas and other technologies have been implemented in proximity labeling approaches, engineered to target specific mRNAs, and then label nearby mRBPs [13]. Using such approaches in studies of the cell cycle against specific mRNAs of interest will be needed to understand how mRBPs regulate the expression of specific mRNAs in the cell cycle. Combining these mRNA-centric approaches with the established mRBP-centric ones will enable the identification of biologically relevant mRNA:mRBP pairs. With such tools at hand, one can be optimistic for rapid progress in understanding how mRBPs control cell division.

Glossary

Budding index

the fraction of cells that are budded in an asynchronous culture of budding yeast cells. The appearance of a bud on the cell surface coincides with the initiation of DNA replication in this organism. Hence, the budding index provides a convenient, morphological readout of the percentage of cells not in the G1 phase of the cell cycle.

Cas

a family of proteins that recognize specific sequences in DNA (e.g., Cas9) or RNA (e.g., Cas13). The sequences are typically found in viruses of prokaryotes. They are composed of clustered regularly interspaced short palindromic repeats (CRISPRs). The host prokaryotic cell uses its Cas systems to detect and destroy DNA from viruses that carry CRISPR sequences. In recent years, engineered Cas systems have been introduced widely in eukaryotes for genome targeting and editing applications.

Cdk

a family of protein serine/threonine kinases that control cell-cycle transitions in all eukaryotes. A Cdk monomer is inactive unless bound to a regulatory protein called a cyclin. Cells typically express several cyclins, and each Cdk/cyclin heterodimer is active at distinct points in the cell cycle. Phosphorylations, both activating and inactivating, further regulate Cdk activity. Association with other proteins, such as inhibitors and processivity factors, adds more layers of Cdk activity control.

P bodies

cytoplasmic processing (P) condensates or aggregates which are not surrounded by a lipid membrane. They contain proteins and mRNAs. Sometimes they are referred to as stress granules. The mRNAs found in a P body are not engaged in translation. Hence, P bodies serve as control points in gene expression.

Proximity labeling

methods that rely on bringing in a living cell an engineered labeling enzyme close to a biomolecule of interest. The labeling enzyme then chemically attaches a tag (usually biotin) to any other biomolecule nearby. The labeled biomolecules are selectively isolated, based on their attached label, from all other components in the cell, and are identified by downstream analytical approaches such as mass spectrometry.