A Novel Long Non-coding RNA, durga Modulates Dendrite Density and Expression of kalirin in Zebrafish

Abstract

Kalirin, a key player in axonal development, nerve growth and synaptic re-modeling, is implicated in many pathological conditions like schizophrenia and autism-spectrum disorders. Alternative promoters and splicing lead to functionally distinct isoforms, but the post-transcriptional regulation of Kalirin has not been studied. Here, we report a novel non-coding RNA, which we name durga, arising from the first exon of kalirin a (kalrna) in the antisense orientation in zebrafish. The kalrna and durga transcripts are barely detectable during early development, but steadily increase by 24 hours post-fertilization (hpf) as the brain develops. Over-expression of durga in the zebrafish embryo led to an increase in kalrna expression. The morphology of the neurons cultured from durga injected embryos had significantly fewer and shorter dendrites. Although durga has no apparent sequence homolog in mammals, based on gene synteny, we found a non-coding RNA arising from the 5' end of the human Kalrn gene and expressed in the human neuronal cell line, SH-SY5Y. We propose that the zebrafish lncRNA durga maintains dendritic length and density through regulation of kalrna expression and this may have further implications in mammalian systems.

Keywords: Kalirin; dendritic morphology; long non-coding RNA; primary neuron culture; zebrafish.

Figure 1

Genomic organization of zebrafish kalrn in zebrafish genome version 9 (Zv9) and zebrafish genome version 10 (Zv10) genome assembly. (A) Pictorial representation and UCSC genome browser tracks of gene re-arrangement of kalrna, mylka and durga in the Zv9 and Zv10 genome assembly. (B) Primers are shown with their position and orientation. Representation not to scale. (C) Expression of durga (137 bp) and novel first exon of kalrna (211 bp) was confirmed by polymerase chain reaction (PCR) with cDNA synthesized from strand specific primer and zebrafish brain RNA.

Figure 2

Developmental expression profile of durga and kalrna in zebrafish. (A) Expression of durga and kalrna was checked by semi-quantitative PCR (A,D respectively), qPCR (B,E respectively) and in situ hybridization (C,F respectively) during 1cell, High, Shield, 2Somites, 30Somites stages. Data shown represent more than three independent experiments. p-values were calculated using Student’s t-test. **p ≤ 0.01, *p ≤ 0.05.

Figure 3

Over-expression of durga enhances kalrna expression. In vitro transcribed durga was injected in one cell stage of zebrafish embryo and over-expression was confirmed by qPCR (A) and in situ hybridization (B) at 2Somites and 30Somites stages. Increase in kalrna expression was seen in qPCR (C) and in situ hybridization (D) at 2Somites and 30Somites stages. Data represent three independent experiments. p-value was calculated using Student’s t-test. *p ≤ 0.05. OE denotes overexpression. N is number of biological replicates.

Figure 4

Over-expression of durga in zebrafish embryos alters dendritic morphology in primary culture of neurons. Over-expression of durga and kalrna was confirmed by qPCR in primary culture of neurons (A). Confocal image of durga over-expressed neuron showed reduced average dendrite number (B,C) and dendrite length (B,D). Data shown represent minimum three independent experiments. p-values were calculated using Student’s t-test. *p < 0.05. OE denotes overexpression.

Figure 5

Presence of non-coding RNA at 5′ end of human Karln. Pictorial representation and UCSC genome browser tracks of non-coding (NR_028136.2) RNA located at 5′ end of human Kalrn. Representation is not to scale (A). Expression of non-coding RNA (NR_028136.2) located at 5′ end of human Kalrn was confirmed by PCR in SH-SY5Y cell line (B).