A growing number of long nuclear-retained non-coding RNAs (ncRNAs) have recently been described. However, few functions have been elucidated for these ncRNAs. Here, we have characterized the function of one such ncRNA, identified as metastasis-associated lung adenocarcinoma transcript 1 (Malat1). Malat1 RNA is expressed in numerous tissues and is highly abundant in neurons. It is enriched in nuclear speckles only when RNA polymerase II-dependent transcription is active. Knock-down studies revealed that Malat1 modulates the recruitment of SR family pre-mRNA-splicing factors to the transcription site of a transgene array. DNA microarray analysis in Malat1- depleted neuroblastoma cells indicates that Malat1 controls the expression of genes involved not only in nuclear processes, but also in synapse function. In cultured hippocampal neurons, knock-down of Malat1 decreases synaptic density, whereas its over-expression results in a cellautonomous increase in synaptic density. Our results suggest that Malat1 regulates synapse formation by modulating the expression of genes involved in synapse formation and/or maintenance.
Introduction
A large portion of the eukaryotic genome is transcribed as non-coding RNAs (ncRNAs) of various sizes ranging from B20 nucleotides to B100 kb (reviewed in Mercer et al, 2009; Wilusz et al, 2009). Despite the increasing number of long ncRNAs (lncRNAs), very few have thus far been assigned a specific function (for review, see Mercer et al, 2009). Whether some of these ncRNAs represent transcriptional noise or are involved in important cellular functions remains a matter of debate (Mattick and Makunin, 2006). However, several observations support the assertion that lncRNAs are likely to have functions in cells (Chamberlain and Brannan, 2001; Willingham et al, 2005; Feng et al, 2006; Mancini-Dinardo et al, 2006; Martianov et al, 2007; Rinn et al, 2007; Yang and Kuroda, 2007; Hirota et al, 2008; Mariner et al, 2008; Nagano et al, 2008; Wang et al, 2008; Yu et al, 2008; Chen and Carmichael, 2009; Clemson et al, 2009; Khalil et al, 2009; Mallik and Lakhotia, 2009; Sasaki et al, 2009; Sunwoo et al, 2009).
Synaptic density analysis
The modification of Malat1 ncRNA level in transfected neuronal cultured cells was first examined by ISH (Supplementary Figure 7). Analysis of synapsin I puncta density was then performed on the same neurons. Punctae of synapsin immunoreactivity were counted along three primary dendrites for each neuron using Metamorph Software. We chose synapsin I immunoreactivity as this accurately correlates with synapse formation (Fletcher et al, 1991; Zhang and Benson, 2001; Graf et al, 2004). Three independent cultures were examined and 10 neurons per condition and per culture were counted. Analyses were performed blind. Two-way ANOVA was used for statistical analysis.
