Evolutionarily Conserved Minor Spliceosome is Required for Differentiating Mouse Retinal Neurons

Abstract

 

Purpose

 

Splicing removes introns and fuses exons, which is essential for eukaryotic gene expression and is carried out by the major spliceosome. The major spliceosome consists of a core set of snRNAs including, U1, U2, U4, U5 and U6 that are required for splicing. Interestingly, in eukaryotes, there exists another spliceosome called the minor spliceosome that is evolutionarily conserved and consists of snRNAs including, U11, U12, U4atac and U6atac. Named thusly, for it removes introns in only 3% of the genes. Given this small subset of introns that it regulates, we wanted to address the following questions. 1) Are the minor spliceosome associated snRNAs expressed in the developing retina? 2) Is the minor spliceosome function required for retinal development?

 

Methods

 

We determined the expression of the minor spliceosome associated snRNAs by RT-PCR and in situ. We also employed P0 in vivo retinal electroporation to inactivate U12 snRNA.

 

Results

 

To test the presence of a functioning spliceosome in the developing retina we examined the expression of U11 and U12 snRNA. Surprisingly, U11 and U12 snRNAs were enriched in newly differentiating neurons, but absent in progenitor cells. Expression of U11 and U12 was observed as distinct puncta in the nuclei of retinal ganglion cells at P0, followed by amacrines and P4 and later in the ONL at P10 and P14. Also, within the nuclei of the same cell, U11 and U12 snRNAs do not overlap, which is surprising since they are thought to work as a di-snRNP. This might suggest that in the retinae, they function independently. Finally, inactivation of U12 snRNA did not perturb the progenitor cell function, but it led to death of terminally differentiating neurons. Specifically, the neuronal death progressed in the order in which they were differentiating. For example, amacrine cell death preceded the rod photoreceptor death.

 

Conclusions

 

The minor spliceosome components such as U11 and U12 are expressed in terminally differentiating retinal neurons. Also, U12 snRNA is required for terminally differentiating retinal neurons postnatally, but is not expressed or required for progenitor cell survival.

 

 

Double Fluorescent in situ for U11 (red) and U12 (green).

 

 

In situ (purple) for U12 snRNA and TUNEL+ (red) cells in P10 and P14 retina after knockdown of U12 snRNA. Cells are marked with GFP (green)

 

Keywords: 698 retinal development • 500 differentiation • 449 cell survival