An official website of the United States government
Structures in the 5′ untranslated regions (UTRs) of mRNAs can physically modulate translation efficiency by impeding the scanning ribosome or by sequestering the translational start site. We assessed the impact of stable protein binding in 5′- and 3′-UTRs on translation efficiency by targeting the MS2 coat protein to a reporter RNA via its hairpin recognition site. Translation was assessed from the reporter RNA when coexpressed with MS2 coat proteins of varying affinities for the RNA, and at different expression levels. Binding of high-affinity proteins in the 5′-UTR hindered translation, whereas no effect was observed when the coat protein was targeted to the 3′-UTR. Inhibition of translation increased with coat protein concentration and affinity, reaching a maximum of 50%–70%. MS2 proteins engineered to bind two reporter mRNA sites had a stronger effect than those binding a single site. Our findings demonstrate that protein binding in an mRNA 5′-UTR physically impedes translation, with the effect governed by affinity, concentration, and sterics.
more »
« less
High-resolution profiling reveals coupled transcriptional and translational regulation of transgenes
Abstract Concentrations of RNAs and proteins provide important determinants of cell fate. Robust gene circuit design requires an understanding of how the combined actions of individual genetic components influence both messenger RNA (mRNA) and protein levels. Here, we simultaneously measure mRNA and protein levels in single cells using hybridization chain reaction Flow-FISH (HCR Flow-FISH) for a set of commonly used synthetic promoters. We find that promoters generate differences in both the mRNA abundance and the effective translation rate of these transcripts. Stronger promoters not only transcribe more RNA but also show higher effective translation rates. While the strength of the promoter is largely preserved upon genome integration with identical elements, the choice of polyadenylation signal and coding sequence can generate large differences in the profiles of the mRNAs and proteins. We used long-read direct RNA sequencing to define the transcription start and splice sites of common synthetic promoters and independently vary the defined promoter and 5′ UTR sequences in HCR Flow-FISH. Together, our high-resolution profiling of transgenic mRNAs and proteins offers insight into the impact of common synthetic genetic components on transcriptional and translational mechanisms. By developing a novel framework for quantifying expression profiles of transgenes, we have established a system for building more robust transgenic systems.
more »
« less
- Award ID(s):
- 2339986
- PAR ID:
- 10610127
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 53
- Issue:
- 11
- ISSN:
- 0305-1048
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
null (Ed.)The Neurospora crassa GUL-1 is part of the COT-1 pathway, which plays key roles in regulating polar hyphal growth and cell wall remodeling. We show that GUL-1 is a bona fide RNA-binding protein (RBP) that can associate with 828 “core” mRNA species. When cell wall integrity (CWI) is challenged, expression of over 25% of genomic RNA species are modulated (2,628 mRNAs, including the GUL-1 mRNA). GUL-1 binds mRNAs of genes related to translation, cell wall remodeling, circadian clock, endoplasmic reticulum (ER), as well as CWI and MAPK pathway components. GUL-1 interacts with over 100 different proteins, including stress-granule and P-body proteins, ER components and components of the MAPK, COT-1, and STRIPAK complexes. Several additional RBPs were also shown to physically interact with GUL-1. Under stress conditions, GUL-1 can localize to the ER and affect the CWI pathway—evident via altered phosphorylation levels of MAK-1, interaction with mak-1 transcript, and involvement in the expression level of the transcription factor adv-1 . We conclude that GUL-1 functions in multiple cellular processes, including the regulation of cell wall remodeling, via a mechanism associated with the MAK-1 pathway and stress-response.more » « less
-
Abstract Poly(A)-binding protein (Pab1 in yeast) is involved in mRNA decay and translation initiation, but its molecular functions are incompletely understood. We found that auxin-induced degradation of Pab1 reduced bulk mRNA and polysome abundance in WT but not in a mutant lacking the catalytic subunit of decapping enzyme (Dcp2), suggesting that enhanced decapping/degradation is a major driver of reduced translation at limiting Pab1. An increased median poly(A) tail length conferred by Pab1 depletion was likewise not observed in the dcp2Δ mutant, suggesting that mRNA isoforms with shorter tails are preferentially decapped/degraded at limiting Pab1. In contrast to findings on mammalian cells, the translational efficiencies (TEs) of many mRNAs were altered by Pab1 depletion; however, these changes were diminished in dcp2Δ cells, suggesting that reduced mRNA abundance is also a major driver of translational reprogramming at limiting Pab1. Thus, assembly of the closed-loop mRNP via PABP–eIF4G interaction appears to be dispensable for wild-type translation of most transcripts at normal mRNA levels. Interestingly, histone mRNAs and proteins were preferentially diminished on Pab1 depletion in DCP2 but not dcp2Δ cells, accompanied by activation of internal cryptic promoters in the manner expected for reduced nucleosome occupancies, implicating Pab1 in post-transcriptional control of histone gene expression.more » « less
-
Protein translation is globally downregulated under stress conditions. Many proteins that are synthesized under stress conditions use a cap-independent translation initiation pathway. A subset of cellular mRNAs that encode for these proteins contain stable secondary structures within their 5′UTR, and initiate cap-independent translation using elements called cap-independent translation enhancers or internal ribosome entry sites within their 5′UTRs. The interaction among initiation factors such as eukaryotic initiation factor 4E (eIF4E), eIF4A, and eIF4GI, especially in regulating the eIF4F complex during noncanonical translation initiation of different 5′UTR mRNAs, is poorly understood. Here, equilibrium-binding assays, CD studies and in vitro translation assays were used to elucidate the recruitment of these initiation factors to the highly structured 5′UTRs of fibroblast-growth factor 9 (FGF-9) and hypoxia inducible factor 1 subunit alpha (HIF-1α) encoding mRNAs. We showed that eIF4A and eIF4E enhanced eIF4GI’s binding affinity to the uncapped 5′UTR of HIF-1α mRNA, inducing conformational changes in the protein/RNA complex. In contrast, these factors have no effect on the binding of eIF4GI to the 5′UTR of FGF-9 mRNA. Recently, Izidoro et al. reported that the interaction of 42nt unstructured RNA to human eIF4F complex is dominated by eIF4E and ATP-bound state of eIF4A. Here, we show that structured 5′UTR mRNA binding mitigates this requirement. Based on these observations, we describe two possible cap-independent translation mechanisms for FGF-9 and HIF-1α encoding mRNAs used by cells to mitigate cellular stress conditions.more » « less
-
Agrobacterium effector protein VirE2 is important for plant transformation. VirE2 likely coats transferred DNA (T-DNA) in the plant cell and protects it from degradation. VirE2 localizes to the plant cytoplasm and interacts with several host proteins. Plant-expressed VirE2 can complement a virE2 mutant Agrobacterium strain to support transformation. We investigated whether VirE2 could facilitate transformation from a nuclear location by affixing to it a strong nuclear localization signal (NLS) sequence. Only cytoplasmic-, but not nuclear-localized, VirE2 could stimulate transformation. To investigate the ways VirE2 supports transformation, we generated transgenic Arabidopsis plants containing a virE2 gene under the control of an inducible promoter and performed RNA-seq and proteomic analyses before and after induction. Some differentially expressed plant genes were previously known to facilitate transformation. Knockout mutant lines of some other VirE2 differentially expressed genes showed altered transformation phenotypes. Levels of some proteins known to be important for transformation increased in response to VirE2 induction, but prior to or without induction of their corresponding mRNAs. Overexpression of some other genes whose proteins increased after VirE2 induction resulted in increased transformation susceptibility. We conclude that cytoplasmically localized VirE2 modulates both plant RNA and protein levels to facilitate transformation.more » « less
