Roles of Plant Glycine-Rich RNA-Binding Proteins in Development and Stress Responses

In recent years, much progress has been made in elucidating the functional roles of plant glycine-rich RNA-binding proteins (GR-RBPs) during development and stress responses. Canonical GR-RBPs contain an RNA recognition motif (RRM) or a cold-shock domain (CSD) at the N-terminus and a glycine-rich domain at the C-terminus, which have been associated with several different RNA processes, such as alternative splicing, mRNA export and RNA editing. However, many aspects of GR-RBP function, the targeting of their RNAs, interacting proteins and the consequences of the RNA target process are not well understood. Here, we discuss recent findings in the field, newly defined roles for GR-RBPs and the actions of GR-RBPs on target RNA metabolism.

Assessing nucleic acid binding activity of four dinoflagellate cold shock domain proteins from Symbiodinium kawagutii and Lingulodinium polyedra

Background Dinoflagellates have a generally large number of genes but only a small percentage of these are annotated as transcription factors. Cold shock domain (CSD) containing proteins (CSPs) account for roughly 60% of these. CSDs are not prevalent in other eukaryotic lineages, perhaps suggesting a lineage-specific expansion of this type of transcription factors in dinoflagellates, but there is little experimental data to support a role for dinoflagellate CSPs as transcription factors. Here we evaluate the hypothesis that dinoflagellate CSPs can act as transcription factors by binding double-stranded DNA in a sequence dependent manner. Results We find that both electrophoretic mobility shift assay (EMSA) competition experiments and selection and amplification binding (SAAB) assays indicate binding is not sequence specific for four different CSPs from two dinoflagellate species. Competition experiments indicate all four CSPs bind to RNA better than double-stranded DNA. Conclusion Dinoflagellate CSPs do not share the nucleic acid binding properties expected for them to function as bone fide transcription factors. We conclude the transcription factor complement of dinoflagellates is even smaller than previously thought suggesting that dinoflagellates have a reduced dependance on transcriptional control compared to other eukaryotes.

Genes with Cold Shock Domain from Eutrema salsugineum (Pall.) for Generating a Cold Stress Tolerance in Winter Rape (Brassica napus L.) Plants

The increased demand in vegetable oil for food purposes and high-protein feed for livestock and poultry encourages producers to expand the production of various oil crops, while occupying rather cold agroclimatic zones. Improved cold and frost resistance of cultivated crops would significantly increase the yield and expand the range of rape cultivation in a number of cold climate regions. Nine transgenic lines of winter rape containing genes encoding proteins with a cold shock domain (CspA и EsCSDP3) were obtained as a result of Agrobacterium transformation. In total, 260 explants were involved in transformation of rape using pBI121-CSPA-plant, with a transformation efficiency of 2.3%; among 750 explants using the pBI-EsCSDP3 construction, the efficiency was 0.4%. As a result of the studies, it was shown that the expression of the new gene Escsdp3 from the plant of Eutrema salsugineum was able to increase the cold and frost resistance of plants as effectively as the cspa gene from E. coli, which is classically used for this purpose. The cold resistance analysis of T1 transgenic plants generation revealed four cold resistant winter rape lines (three lines with the cspA-plant gene and one line with the Escsdp3 gene). The transfer of Escsdp3 and cspA-plant genes into winter rape plants led to a significant increase in frost resistance of plants. Two winter rapeseed lines were resistant to freezing (with the cspA-plant gene and with the Escsdp3 gene). Non-hardened transgenic plants remained viable after 24 h of exposure to negative temperatures up to −5 °C, and plants that passed through the hardening stage survived after freezing at −16 °C.

Oncolytic virotherapy induced CSDE1 neo-antigenesis restricts VSV replication but can be targeted by immunotherapy

In our clinical trials of oncolytic vesicular stomatitis virus expressing interferon beta (VSV-IFNβ), several patients achieved initial responses followed by aggressive relapse. We show here that VSV-IFNβ-escape tumors predictably express a point-mutated CSDE1P5S form of the RNA-binding Cold Shock Domain-containing E1 protein, which promotes escape as an inhibitor of VSV replication by disrupting viral transcription. Given time, VSV-IFNβ evolves a compensatory mutation in the P/M Inter-Genic Region which rescues replication in CSDE1P5S cells. These data show that CSDE1 is a major cellular co-factor for VSV replication. However, CSDE1P5S also generates a neo-epitope recognized by non-tolerized T cells. We exploit this predictable neo-antigenesis to drive, and trap, tumors into an escape phenotype, which can be ambushed by vaccination against CSDE1P5S, preventing tumor escape. Combining frontline therapy with escape-targeting immunotherapy will be applicable across multiple therapies which drive tumor mutation/evolution and simultaneously generate novel, targetable immunopeptidomes associated with acquired treatment resistance.

Lin28, a major translation reprogramming factor, gains access to YB-1-packaged mRNA through its cold-shock domain

The RNA-binding protein Lin28 (Lin28a) is an important pluripotency factor that reprograms translation and promotes cancer progression. Although Lin28 blocks let-7 microRNA maturation, Lin28 also binds to a large set of cytoplasmic mRNAs directly. However, how Lin28 regulates the processing of many mRNAs to reprogram global translation remains unknown. We show here, using a structural and cellular approach, a mixing of Lin28 with YB-1 (YBX1) in the presence of mRNA owing to their cold-shock domain, a conserved β-barrel structure that binds to ssRNA cooperatively. In contrast, the other RNA binding-proteins without cold-shock domains tested, HuR, G3BP-1, FUS and LARP-6, did not mix with YB-1. Given that YB-1 is the core component of dormant mRNPs, a model in which Lin28 gains access to mRNPs through its co-association with YB-1 to mRNA may provide a means for Lin28 to reprogram translation. We anticipate that the translational plasticity provided by mRNPs may contribute to Lin28 functions in development and adaptation of cancer cells to an adverse environment.

Cold-Shock Domains—Abundance, Structure, Properties, and Nucleic-Acid Binding

The cold-shock domain has a deceptively simple architecture but supports a complex biology. It is conserved from bacteria to man and has representatives in all kingdoms of life. Bacterial cold-shock proteins consist of a single cold-shock domain and some, but not all are induced by cold shock. Cold-shock domains in human proteins are often associated with natively unfolded protein segments and more rarely with other folded domains. Cold-shock proteins and domains share a five-stranded all-antiparallel β-barrel structure and a conserved surface that binds single-stranded nucleic acids, predominantly by stacking interactions between nucleobases and aromatic protein sidechains. This conserved binding mode explains the cold-shock domains’ ability to associate with both DNA and RNA strands and their limited sequence selectivity. The promiscuous DNA and RNA binding provides a rationale for the ability of cold-shock domain-containing proteins to function in transcription regulation and DNA-damage repair as well as in regulating splicing, translation, mRNA stability and RNA sequestration.