mRNA Decapping Research Articles

The length scale of multivalent interactions is evolutionarily conserved in fungal and vertebrate phase-separating proteins.

One key feature of proteins that form liquid droplets by phase separation inside a cell is multivalency-the presence of multiple sites that mediate interactions with other proteins. We know little about the variation of multivalency on evolutionary time scales. Here, we investigated the long-term evolution (∼600 million years) of multivalency in fungal mRNA decapping subunit 2 protein (Dcp2), and in the FET (FUS, EWS and TAF15) protein family. We found that multivalency varies substantially among the orthologs of these proteins. However, evolution has maintained the length scale at which sequence motifs that enable protein-protein interactions occur. That is, the total number of such motifs per hundred amino acids is higher and less variable than expected by neutral evolution. To help explain this evolutionary conservation, we developed a conformation classifier using machine-learning algorithms. This classifier demonstrates that disordered segments in Dcp2 and FET proteins tend to adopt compact conformations, which is necessary for phase separation. Thus, the evolutionary conservation we detected may help proteins preserve the ability to undergo phase separation. Altogether, our study reveals that the length scale of multivalent interactions is an evolutionarily conserved feature of two classes of phase-separating proteins in fungi and vertebrates.

The Mimivirus L375 Nudix enzyme hydrolyzes the 5' mRNA cap.

The giant Mimivirus is a member of the nucleocytoplasmic large DNA viruses (NCLDV), a group of diverse viruses that contain double-stranded DNA (dsDNA) genomes that replicate primarily in eukaryotic hosts. Two members of the NCLDV, Vaccinia Virus (VACV) and African Swine Fever Virus (ASFV), both synthesize Nudix enzymes that have been shown to decap mRNA, a process thought to accelerate viral and host mRNA turnover and promote the shutoff of host protein synthesis. Mimivirus encodes two Nudix enzymes in its genome, denoted as L375 and L534. Importantly, L375 exhibits sequence similarity to ASFV-DP and eukaryotic Dcp2, two Nudix enzymes shown to possess mRNA decapping activity. In this work, we demonstrate that recombinant Mimivirus L375 cleaves the 5’ m7GpppN mRNA cap, releasing m7GDP as a product. L375 did not significantly cleave mRNAs containing an unmethylated 5’GpppN cap, indicating that this enzyme specifically hydrolyzes methylated-capped transcripts. A point mutation in the L375 Nudix motif completely eliminated cap hydrolysis, showing that decapping activity is dependent on this motif. Addition of uncapped RNA significantly reduced L375 decapping activity, suggesting that L375 may recognize its substrate through interaction with the RNA body.

The mRNA decapping protein 2 (DCP2) is a major regulator of developmental events in Drosophila-insights from expression paradigms.

The Drosophila genome codes for two decapping proteins, DCP1 and DCP2, out of which DCP2 is the active decapping enzyme. The present endeavour explores the endogenous promoter firing, transcript and protein expression of DCP2 in Drosophila wherein, besides a ubiquitous expression across development, we identify an active expression paradigm during dorsal closure and a plausible moonlighting expression in the Corazonin neurons of the larval brain. We also demonstrate that the ablation of DCP2 leads to embryonic lethality and defects in vital morphogenetic processes whereas a knockdown of DCP2 in the Corazonin neurons reduces the sensitivity to ethanol in adults, thereby ascribing novel regulatory roles to DCP2. Our findings unravel novel putative roles for DCP2 and identify it as a candidate for studies on the regulated interplay of essential molecules during early development in Drosophila, nay the living world.

Exploring the role of RRM domains and conserved aromatic residues in RGG motif of eIF4G-binding translation repressor protein Sbp1.

Background:RNA binding proteins play crucial role in determining if a given mRNA will be translated, stored, or degraded. Sbp1 is an RGG-motif containing protein that is implicated in affecting mRNA decapping and translation. Sbp1 represses translation by binding eIF4G1 through its RGG-motif and activates decapping when overexpressed. In this report, we have assessed the genetic interaction of Sbp1 with decapping activators such as Dhh1, Pat1, and Scd6. We have further analyzed the importance of different domains and specific conserved residues of Sbp1 inits ability to cause over-expression mediated growth defect. Method:Sequence alignment was performed to identify conserved aromatic residues to be mutated. Using site-directed mutagenesis several point mutations and domain deletions were created in Sbp1 expressed under a galactose-inducible promoter. The mutants were tested for their ability to cause growth defect upon over-expression. The ability of Sbp1 to affect over-expression mediated growth defect of other decapping activators was tested using growth assay. Live cell imaging was done to study localization of Sbp1 and its RRM-deletion mutants to RNA granules upon glucose starvation. Results:Mutation of several aromatic residues in the RGG-motif and that of the phosphorylation sites in the RRM domain of Sbp1 did not affect the growth defect phenotype. Deletion of another eIF4G1-binding RGG-motif protein Scd6 does not affect the ability of Sbp1 to cause growth defect. Moreover, absence of Sbp1 did not affect the growth defect phenotypes observed upon overexpression of decapping activators Dhh1 and Pat1. Strikingly deletion of both the RRM domains (RRM1 and RRM2) and not the RNP motifs within them compromised the growth defect phenotype. Sbp1 mutant lacking both RRM1 and RRM2 was highly defective in localizing to RNA granules. Conclusion:This study identifies an important role of RRM domains independent of the RNP motif in Sbp1 function.

The mRNA decapping complex is buffered by nuclear localization.

mRNA decay is a key step in regulating the cellular proteome. Processing bodies (P-bodies) are thought to be sites of mRNA decay and/or storage. P-body units assemble into P-body granules under stress conditions. How this assembly is regulated, however, remains poorly understood. Here, we show, in the yeast Saccharomyces cerevisiae, that the translational repressor Scd6 and the decapping stimulator Edc3 act partially redundantly in P-body assembly by sequestering the Dcp1-Dcp2 (denoted Dcp1/2) decapping complex in the cytoplasm and preventing it from becoming imported into the nucleus by the karyopherin β protein Kap95. One of two nuclear localization signals in Dcp2 overlaps with the RNA-binding site, suggesting an additional mechanism to regulate Dcp1/2 localization. Nuclear Dcp1/2 does not drive mRNA decay and might be stored there as a readily releasable pool, indicating a dynamic equilibrium between cytoplasmic and nuclear Dcp1/2. Cytoplasmic Dcp1/2 is linked to Dhh1 via Edc3. Functional P-bodies are present at the endoplasmic reticulum where Dcp2 potentially acts to increase the local concentration of Dhh1 through interaction with Edc3 to drive phase separation and hence P-body formation.

MRNA-Decapping Associated DcpS Enzyme Controls Critical Steps of Neuronal Development.

Homozygous mutations in the gene encoding the scavenger mRNA-decapping enzyme, DcpS, have been shown to underlie developmental delay and intellectual disability. Intellectual disability is associated with both abnormal neocortical development and mRNA metabolism. However, the role of DcpS and its scavenger decapping activity in neuronal development is unknown. Here, we show that human neurons derived from patients with a DcpS mutation have compromised differentiation and neurite outgrowth. Moreover, in the developing mouse neocortex, DcpS is required for the radial migration, polarity, neurite outgrowth, and identity of developing glutamatergic neurons. Collectively, these findings demonstrate that the scavenger mRNA decapping activity contributes to multiple pivotal roles in neural development and further corroborate that mRNA metabolism and neocortical pathologies are associated with intellectual disability.

Is mRNA decapping by ApaH like phosphatases present in eukaryotes beyond the Kinetoplastida?

BackgroundApaH like phosphatases (ALPHs) originate from the bacterial ApaH protein and have been identified in all eukaryotic super-groups. Only two of these proteins have been functionally characterised. We have shown that the ApaH like phosphatase ALPH1 from the Kinetoplastid Trypanosoma brucei is the mRNA decapping enzyme of the parasite. In eukaryotes, Dcp2 is the major mRNA decapping enzyme and mRNA decapping by ALPHs is unprecedented, but the bacterial ApaH protein was recently found decapping non-conventional caps of bacterial mRNAs. These findings prompted us to explore whether mRNA decapping by ALPHs is restricted to Kinetoplastida or could be more widespread among eukaryotes.ResultsWe screened 827 eukaryotic proteomes with a newly developed Python-based algorithm for the presence of ALPHs and used the data to characterize the phylogenetic distribution, conserved features, additional domains and predicted intracellular localisation of this protein family. For most organisms, we found ALPH proteins to be either absent (495/827 organisms) or to have non-cytoplasmic localisation predictions (73% of all ALPHs), excluding a function in mRNA decapping. Although, non-cytoplasmic ALPH proteins had in vitro mRNA decapping activity. Only 71 non-Kinetoplastida have ALPH proteins with predicted cytoplasmic localisations. However, in contrast to Kinetoplastida, these organisms also possess a homologue of Dcp2 and in contrast to ALPH1 of Kinetoplastida, these ALPH proteins are very short and consist of the catalytic domain only.ConclusionsALPH was present in the last common ancestor of eukaryotes, but most eukaryotes have either lost the enzyme, or use it exclusively outside the cytoplasm. The acceptance of mRNA as a substrate indicates that ALPHs, like bacterial ApaH, have a wide substrate range: the need to protect mRNAs from unregulated degradation is one possible explanation for the selection against the presence of cytoplasmic ALPH proteins in most eukaryotes. Kinetoplastida succeeded to exploit ALPH as their only or major mRNA decapping enzyme. 71 eukaryotic organisms outside the Kinetoplastid lineage have short ALPH proteins with cytoplasmic localisation predictions: whether these proteins are used as decapping enzymes in addition to Dcp2 or else have adapted to not accept mRNAs as a substrate, remains to be explored.

MRNA decapping factor Dcp1a is essential for embryonic growth in mice

mRNA decapping is a critical step in posttranscriptional regulation of gene expression in eukaryotes. Although Dcp1a is a well characterized and widely conserved mRNA decapping factor, little is known about its physiological function. To extend our understanding of Dcp1a function in vivo, we employed a transgenic rescue strategy to produce Dcp1a-deficient mice using the CRISPR/Cas9 system. This approach arrowed us to generate heterozygous Dcp1a mice and define the phenotype of Dcp1a-deficient embryos. We found that expression of Dcp1a protein, which is detectable in most mouse tissues, was developmentally regulated through embryonic growth, and that depletion of the Dcp1a gene resulted in embryonic lethality around embryonic day 10.5 (E10.5) concomitant with massive growth retardation and cardiac developmental defects. Moreover, the embryonic lethality was fully rescued by transgenic expression of exogenous human Dcp1a. Together, our results suggest that Dcp1a is required for embryonic growth.

Prognostic value and potential molecular mechanism of the like-Sm gene family in early-stage pancreatic ductal adenocarcinoma.

BackgroundThe purpose of this study was to investigate the prognostic significance of like-Sm (LSM) genes in early pancreatic ductal adenocarcinoma (PDAC) and explore the potential molecular mechanism. The protein product of the LSM1 gene is also known as CASM and YJL124C, while that of the LSM4 gene is known as GRP and YER112W.MethodsData from 112 patients attached to the Whipple surgery were collected from the TCGA database of clinical characteristics and survival data. The Kaplan-Meier method and the multivariate Cox proportional risk regression model were used to analyze the impact of LSM genes on outcomes in these 112 patients. We performed gene-gene interaction (GGI) and protein-protein interaction (PPI) analysis to probe interactions between LSM family genes. Bioinformatics techniques were applied to study the potential early-stage molecular mechanisms of LSM genes. Previously, only a few studies have explored the role and potential mechanisms of LSM1 in pancreatic tumor transformation, revealing possible links to transforming growth factor-β, altering the expression of MMP1, uPAR, and SerpinB5 to enhance invasion and metastasis in pancreatic cancer, and facilitating mRNA decapping and degradation. Gene set enrichment analysis (GSEA) also proved that LSM genes are associated with RNA splicing, RNA synthesis, and RNA decomposition, and they may indirectly cause carcinogenesis through other genes such as myc.ResultsThe results showed that LSM1 (adjusted P=0.004) and LSM4 (adjusted P=0.034) were associated with the prognosis of patients with PDAC, and patients with high expression levels of LSM1 (adjusted HR =2.338) or LSM4 (adjusted HR =1.803) tended to experience bad outcomes.ConclusionsOur study revealed that LSM1 and LSM4 might be used as prognostic biomarkers in early PDAC.

Biomolecular condensates amplify mRNA decapping by biasing enzyme conformation.

Cells organize biochemical processes into biological condensates. P-bodies are cytoplasmic condensates enriched in enzymes important for mRNA degradation and have been identified as sites of both storage and decay. How these opposing outcomes can be achieved in condensates remains unresolved. mRNA decapping immediately precedes degradation and the Dcp1/Dcp2 decapping complex is enriched in P-bodies. Here, we show Dcp1/Dcp2 activity is modulated in condensates and depends on the interactions promoting phase separation. We find Dcp1/Dcp2 phase separation stabilizes an inactive conformation in Dcp2 to inhibit decapping. The activator Edc3 causes a conformational change in Dcp2 and rewires the protein-protein interactions to stimulate decapping in condensates. Disruption of the inactive conformation dysregulates decapping in condensates. Our results indicate regulation of enzymatic activity in condensates relies on a coupling across length scales ranging from microns to Ångstroms. We propose this regulatory mechanism may control the functional state of P-bodies and related phase-separated compartments.

Biological implications of decapping: beyond bulk mRNA decay.

It is well established that mRNA steady-state levels do not directly correlate with transcription rate. This is attributed to the multiple post-transcriptional mechanisms, which control both mRNA turnover and translation within eukaryotic cells. One such mechanism is the removal of the 5' end cap structure of RNAs (decapping). This 5' cap plays a fundamental role in cellular functions related to mRNA processing, transport, translation, quality control, and decay, while its chemical modifications influence the fate of cytoplasmic mRNAs. Decapping is a highly controlled process, performed by multiple decapping enzymes, and regulated by complex cellular networks. In this review, we provide an updated synopsis of 5' end modifications and functions, and give an overview of mRNA decapping enzymes, presenting their enzymatic properties. Focusing on DCP2 decapping enzyme, a major component on the 5'-3' mRNA decay pathway, we describe cis-elements and trans-acting factors that affect its activity, substrate specificity, and cellular localization. Finally, we discuss current knowledge on the biological functions of mRNA decapping and decay factors, highlighting the major questions that remain to be addressed.

EDC3 phosphorylation regulates growth and invasion through controlling P-body formation and dynamics.

Regulation of mRNA stability and translation plays a critical role in determining protein abundance within cells. Processing bodies (P-bodies) are critical regulators of these processes. Here, we report that the Pim1 and 3 protein kinases bind to the P-body protein enhancer of mRNA decapping 3 (EDC3) and phosphorylate EDC3 on serine (S)161, thereby modifying P-body assembly. EDC3 phosphorylation is highly elevated in many tumor types, is reduced upon treatment of cells with kinase inhibitors, and blocks the localization of EDC3 to P-bodies. Prostate cancer cells harboring an EDC3 S161A mutation show markedly decreased growth, migration, and invasion in tissue culture and in xenograft models. Consistent with these phenotypic changes, the expression of integrin β1 and α6 mRNA and protein is reduced in these mutated cells. These results demonstrate that EDC3 phosphorylation regulates multiple cancer-relevant functions and suggest that modulation of P-body activity may represent a new paradigm for cancer treatment.

Translation machinery reprogramming in programmed cell death in Saccharomyces cerevisiae

Programmed cell death involves complex molecular pathways in both eukaryotes and prokaryotes. In Escherichia coli, the toxin–antitoxin system (TA-system) has been described as a programmed cell death pathway in which mRNA and ribosome organizations are modified, favoring the production of specific death-related proteins, but also of a minor portion of survival proteins, determining the destiny of the cell population. In the eukaryote Saccharomyces cerevisiae, the ribosome was shown to change its stoichiometry in terms of ribosomal protein content during stress response, affecting the relative proportion between ohnologs, i.e., the couple of paralogs derived by a whole genome duplication event. Here, we confirm the differential expression of ribosomal proteins in yeast also during programmed cell death induced by acetic acid, and we highlight that also in this case pairs of ohnologs are involved. We also show that there are different trends in cytosolic and mitochondrial ribosomal proteins gene expression during the process. Moreover, we show that the exposure to acetic acid induces the differential expression of further genes coding for products related to translation processes and to rRNA post-transcriptional maturation, involving mRNA decapping, affecting translation accuracy, and snoRNA synthesis. Our results suggest that the reprogramming of the overall translation apparatus, including the cytosolic ribosome reorganization, are relevant events in yeast programmed cell death induced by acetic acid.

Diversity of LSM Family Proteins: Similarities and Differences.

Members of the Lsm protein family are found in all three domains of life: bacteria, archaea, and eukarya. They are involved in numerous processes associated with RNA processing and gene expression regulation. A common structural feature of all Lsm family proteins is the presence of the Sm fold consisting of a five-stranded β-sheet and an α-helix at the N-terminus. Heteroheptameric eukaryotic Sm and Lsm proteins participate in the formation of spliceosomes and mRNA decapping. Homohexameric bacterial Lsm protein, Hfq, is involved in the regulation of transcription of different mRNAs by facilitating their interactions with small regulatory RNAs. Furthermore, recently obtained data indicate a new role of Hfq as a ribosome biogenesis factor, as it mediates formation of the productive structure of the 17S rRNA 3'- and 5'-sequences, facilitating their further processing by RNases. Lsm archaeal proteins (SmAPs) form homoheptamers and likely interact with single-stranded uridine-rich RNA elements, although the role of these proteins in archaea is still poorly understood. In this review, we discuss the structural features of the Lsm family proteins from different life domains and their structure-function relationships.

Pseudomonas syringae pv. syringae Infection Orchestrates the Fate of the Arabidopsis J Domain Containing Cochaperone and Decapping Protein Factor 5

Plants are equipped with numerous defense strategies to combat any biotic or abiotic challenges. Several protein factors are constantly engaged in the surveillance process, thus production of any aberrant messenger RNAs (mRNAs) and proteins are restricted. Eukaryotic mRNAs go through a number of scrutiny processes, whereby several protein components including mRNA decapping factors play a critical role in degrading the non-physiological mRNAs, which would otherwise have detrimental effect when translated. Similarly newly synthesized proteins also pass through quality control process mediated by chaperones and cochaperones harboured within the endoplasmic reticulum, ensuring proper protein folding and minimize the occurrence of protein misfolding. Interestingly, the results of the present study revealed that the decapping protein factor, DCP5 of the Arabidopsis thaliana physically interacts with the J domain-containing cochaperone, ERdj3B in the processing bodies (P-bodies). Upon pathogen infection by the Pseudomonas syringae pv. syringae B728a strain (PssB728a), the ERdj3B was induced for degradation via the 26S proteasome pathway as early as 120 min post infection (mpi). Also under pathogen-challenged conditions, DCP5 relocalizes to the cytoplasm and the number of DCP5-induced P-bodies was significantly reduced at 90 and 120 mpi. The findings of the present investigation highlight the possible significance of this interaction, primarily contributing for maintaining proteostasis and their plausible role during plant defense process.

Suppressors of mRNA Decapping Defects Restore Growth Without Major Effects on mRNA Decay Rates or Abundance.

Faithful degradation of mRNAs is a critical step in gene expression, and eukaryotes share a major conserved mRNA decay pathway. In this major pathway, the two rate-determining steps in mRNA degradation are the initial gradual removal of the poly(A) tail, followed by removal of the cap structure. Removal of the cap structure is carried out by the decapping enzyme, containing the Dcp2 catalytic subunit. Although the mechanism and regulation of mRNA decay is well understood, the consequences of defects in mRNA degradation are less clear. Dcp2 has been reported as either essential or nonessential. Here, we clarify that Dcp2 is not absolutely required for spore germination and extremely slow growth, but in practical terms it is impossible to continuously culture dcp2∆ under laboratory conditions without suppressors arising. We show that null mutations in at least three different genes are each sufficient to restore growth to a dcp2∆, of which kap123∆ and tl(gag)g∆ appear the most specific. We show that kap123∆ and tl(gag)g∆ suppress dcp2 by mechanisms that are different from each other and from previously isolated dcp2 suppressors. The suppression mechanism for tL(GAG)G is determined by the unique GAG anticodon of this tRNA, and thus likely by translation of some CUC or CUU codons. Unlike previously reported suppressors of decapping defects, these suppressors do not detectably restore decapping or mRNA decay to normal rates, but instead allow survival while only modestly affecting RNA homeostasis. These results provide important new insight into the importance of decapping, resolve previously conflicting publications about the essentiality of DCP2, provide the first phenotype for a tl(gag)g mutant, and show that multiple distinct mechanisms can bypass Dcp2 requirement.

The Arabidopsis thaliana mRNA decay factor PAT1 functions in osmotic stress responses and decaps ABA-responsive genes.

mRNA decapping plays essential roles in regulating gene expression during cellular reprogramming in response to developmental and environmental cues. The evolutionarily conserved PAT1 proteins activate decapping by binding mRNA, recruiting other decapping components, and promoting processing body (PB) assembly. Arabidopsis encodes 3 PAT proteins: PAT1, PATH1, and PATH2. Here, we report that only pat1 mutants exhibit hypersensitivity to ABA and that transcripts of ABA-responsive genes, but not those of ABA biosynthesis genes, persist longer in these mutants. The pat1 mutants also exhibit increased resistance to drought stress and resistance to Pythiumirregulare. This is supported by assays showing that PAT1 functions specifically in decapping of the canonical ABA-responsive gene COR15A. In summary, PAT1 protein mediates decay of ABA-responsive genes and, thus, regulates stress responses.

Enhancer of mRNA Decapping protein 4 (EDC4) interacts with replication protein a (RPA) and contributes to Cisplatin resistance in cervical Cancer by alleviating DNA damage

BackgroundCervical cancer (CC) is the third most common gynecological malignancy around the world. Cisplatin is an effective drug, but cisplatin resistance is a vital factor limiting the clinical usage of cisplatin. Enhancer of mRNA decapping protein 4 (EDC4) is a known regulator of mRNA decapping, which was related with genome stability and sensitivity of drugs. This research was to investigate the mechanism of EDC4 on cisplatin resistance in CC. Two human cervical cancer cell lines, HeLa and SiHa, were used to investigate the role of EDC4 on cisplatin resistance in vitro. The knockdown or overexpression of EDC4 or replication protein A (RPA) in HeLa or SiHa cells was performed by transfection. Cell viability was analyzed by MTT assay. The growth of cancer cells was evaluated by colony formation assay. DNA damage was measured by γH2AX (a sensitive DNA damage response marker) immunofluorescent staining. The binding of EDC4 and RPA was analyzed by immunoprecipitation.ResultsEDC4 knockdown in cervical cancer cells (HeLa and SiHa) enhanced cisplatin sensitivity and cisplatin induced cell growth inhibition and DNA damage. EDC4 overexpression reduced DNA damage caused by cisplatin and enhanced cell growth of cervical cancer cells. EDC4 could interact with RPA and promote RPA phosphorylation. RPA knockdown reversed the inhibitory effect of EDC4 on cisplatin-induced DNA damage.ConclusionThe present results indicated that EDC4 is responsible for the cisplatin resistance partly through interacting with RPA in cervical cancer by alleviating DNA damage. This study indicated that EDC4 or RPA may be novel targets to combat chemotherapy resistance in cervical cancer.Graphical abstract

Autoreactivity of Broadly Neutralizing Influenza Human Antibodies to Human Tissues and Human Proteins.

Broadly neutralizing monoclonal antibodies (bNAbs) against conserved domains in the influenza hemagglutinin are in clinical trials. Several next generation influenza vaccines designed to elicit such bNAbs are also in clinical development. One of the common features of the isolated bNAbs is the use of restricted IgVH repertoire. More than 80% of stem-targeting bNAbs express IgVH1-69, which may indicate genetic constraints on the evolution of such antibodies. In the current study, we evaluated a panel of influenza virus bNAbs in comparison with HIV-1 MAb 4E10 and anti-RSV MAb Palivizumab (approved for human use) for autoreactivity using 30 normal human tissues microarray and human protein (>9000) arrays. We found that several human bNAbs (CR6261, CR9114, and F2603) reacted with human tissues, especially with pituitary gland tissue. Importantly, protein array analysis identified high-affinity interaction of CR6261 with the autoantigen “Enhancer of mRNA decapping 3 homolog” (EDC3), which was not previously described. Moreover, EDC3 competed with hemagglutinin for binding to bNAb CR6261. These autoreactivity findings underscores the need for careful evaluation of such bNAbs for therapeutics and stem-based vaccines against influenza virus.

Roles of Dhh1 RNA helicase in yeast filamentous growth: Analysis of N-terminal phosphorylation residues and ATPase domains.

In yeast Saccharomyces cerevisiae, the Dhh1 protein, a member of the DEAD-box RNA helicase, stimulates Dcp2/Dcp1-mediated mRNA decapping and functions as a general translation repressor. Dhh1 also positively regulates translation of a selected set of mRNAs, including Ste12, a transcription factor for yeast mating and pseudohyphal growth. Given the diverse functions of Dhh1, we investigated whether the putative phosphorylation sites or the conserved motifs for the DEAD-box RNA helicases were crucial in the regulatory roles of Dhh1 during pseudohyphal growth. Mutations in the ATPase A or B motif (DHH1-K96R or DHH1-D195A) showed significant defects in pseudohyphal colony morphology and agar invasive phenotypes. The N-terminal phospho-mimetic mutation, DHH1-T16E, showed defects in pseudohyphal phenotypes. Decreased levels of Ste12 protein were also observed in these pseudohyphal-defective mutant cells under filamentous-inducing low nitrogen conditions. We suggest that the ATPase motifs and the Thr16 phosphorylation site of Dhh1 are crucial to its regulatory roles in pseudohyphal growth under low nitrogen conditions.