Alternative LIM homeodomain splice variants are dynamically regulated at key developmental steps in vertebrates.

BACKGROUND: Alternative splicing provides a broad strategy to amplify the genome. Yet how alternative splicing influences neurodevelopment or indeed which variants are translated at developmental choice points remains poorly explored. Here we focused on a gene important for neurodevelopment, the Lim homeodomain transcription factor, Lhx9. Lhx9 has two noncanonical splice variants, Lhx9a and Lhx9b which compared with the canonical variant Lhx9c have a truncated homeodomain and an alternative C-terminal sequence, suggesting that, if translated, these variants could differently impact on cellular function. RESULTS: We created a unique antibody tool designed to selectively detect noncanonical Lhx9 variants (Lhx9ab) and used this to examine the protein expression dynamics in embryos. Lhx9ab variants were translated and dynamically expressed similarly between mouse and chicken at key developmental choice points in the spinal cord, limbs and urogenital ridge. Within the spinal cord, enrichment of Lhx9c vs Lhx9ab expression was observed during key migration and axonal projection choice points. CONCLUSIONS: These data support the notion that the expression dynamics between canonical and noncanonical Lhx9 variants could play an important role in spinal neuron maturation. More broadly, determining the temporal dynamics of alternative protein variants is a key entry point to understand how splicing influences developmental processes.

Robo2 Receptor Gates the Anatomical Divergence of Neurons Derived From a Common Precursor Origin.

Sensory information relayed to the brain is dependent on complex, yet precise spatial organization of neurons. This anatomical complexity is generated during development from a surprisingly small number of neural stem cell domains. This raises the question of how neurons derived from a common precursor domain respond uniquely to their environment to elaborate correct spatial organization and connectivity. We addressed this question by exploiting genetically labeled mouse embryonic dorsal interneuron 1 (dI1) neurons that are derived from a common precursor domain and give rise to spinal projection neurons with distinct organization of cell bodies with axons projecting either commissurally (dI1c) or ipsilaterally (dI1i). In this study, we examined how the guidance receptor, Robo2, which is a canonical Robo receptor, influenced dI1 guidance during embryonic development. Robo2 was enriched in embryonic dI1i neurons, and loss of Robo2 resulted in misguidance of dI1i axons, whereas dI1c axons remained unperturbed within the mantle zone and ventral commissure. Further, Robo2 profoundly influenced dI1 cell body migration, a feature that was partly dependent on Slit2 signaling. These data suggest that dI1 neurons are dependent on Robo2 for their organization. This work integrated with the field support of a model whereby canonical Robo2 vs. non-canonical Robo3 receptor expression facilitates projection neurons derived from a common precursor domain to read out the tissue environment uniquely giving rise to correct anatomical organization.

The decapping activator Edc3 and the Q/N-rich domain of Lsm4 function together to enhance mRNA stability and alter mRNA decay pathway dependence in Saccharomyces cerevisiae.

The rate and regulation of mRNA decay are major elements in the proper control of gene expression. Edc3 and Lsm4 are two decapping activator proteins that have previously been shown to function in the assembly of RNA granules termed P bodies. Here, we show that deletion of edc3, when combined with a removal of the glutamine/asparagine rich region of Lsm4 (edc3Δ lsm4ΔC) reduces mRNA stability and alters pathways of mRNA degradation. Multiple tested mRNAs exhibited reduced stability in the edc3Δ lsm4ΔC mutant. The destabilization was linked to an increased dependence on Ccr4-mediated deadenylation and mRNA decapping. Unlike characterized mutations in decapping factors that either are neutral or are able to stabilize mRNA, the combined edc3Δ lsm4ΔC mutant reduced mRNA stability. We characterized the growth and activity of the major mRNA decay systems and translation in double mutant and wild-type yeast. In the edc3Δ lsm4ΔC mutant, we observed alterations in the levels of specific mRNA decay factors as well as nuclear accumulation of the catalytic subunit of the decapping enzyme Dcp2. Hence, we suggest that the effects on mRNA stability in the edc3Δ lsm4ΔC mutant may originate from mRNA decay protein abundance or changes in mRNPs, or alternatively may imply a role for P bodies in mRNA stabilization.

Membrane-association of mRNA decapping factors is independent of stress in budding yeast.

Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation.

The cytoplasmic mRNA degradation factor Pat1 is required for rRNA processing.

Pat1 is a key cytoplasmic mRNA degradation factor, the loss of which severely increases mRNA half-lives. Several recent studies have shown that Pat1 can enter the nucleus and can shuttle between the nucleus and the cytoplasm. As a result, many nuclear roles have been proposed for Pat1. In this study, we analyzed four previously suggested nuclear roles of Pat1 and show that Pat1 is not required for efficient pre-mRNA splicing or pre-mRNA decay in yeast. However, lack of Pat1 results in accumulation of pre-rRNA processing intermediates. Intriguingly, we identified a novel genetic relationship between Pat1 and the rRNA decay machinery, specifically the exosome and the TRAMP complex. While the pre-rRNA processing intermediates that accumulate in the pat1 deletion mutant are, at least to some extent, recognized as aberrant by the rRNA degradation machinery, it is unlikely that these accumulations are the cause of their synthetic sick relationship. Here, we show that the dysregulation of the levels of mRNAs related to ribosome biogenesis could be the cause of the accumulation of the pre-rRNA processing intermediates. Although our results support a role for Pat1 in transcription, they nevertheless suggest that the primary cause of the dysregulated mRNA levels is most likely due to Pat1's role in mRNA decapping and mRNA degradation.