Natural antisense transcripts and long non-coding RNA in Neurospora crassa

Abstract

The prevalence of long non-coding RNAs (lncRNA) and natural antisense transcripts (NATs) has been reported in a variety of organisms. While a consensus has yet to be reached on their global importance, an increasing number of examples have been shown to be functional, regulating gene expression at the transcriptional and post-transcriptional level. Here, we use RNA sequencing data from the ABI SOLiD platform to identify lncRNA and NATs obtained from samples of the filamentous fungus Neurospora crassa grown under different light and temperature conditions. We identify 939 novel lncRNAs, of which 477 are antisense to annotated genes. Across the whole dataset, the extent of overlap between sense and antisense transcripts is large: 371 sense/antisense transcripts are complementary over 500 nts or more and 236 overlap by more than 1000 nts. Most prevalent are 3' end overlaps between convergently transcribed sense/antisense pairs, but examples of divergently transcribed pairs and nested transcripts are also present. We confirm the expression of a subset of sense/antisense transcript pairs by qPCR. We examine the size, types of overlap and expression levels under the different environmental stimuli of light and temperature, and identify 11 lncRNAs that are up-regulated in response to light. We also find differences in transcript length and the position of introns between protein-coding transcripts that have antisense expression and transcripts with no antisense expression. These results demonstrate the ability of N. crassa lncRNAs and NATs to be regulated by different environmental stimuli and provide the scope for further investigation into the function of NATs.

Figure 1. Properties of novel lncRNAs and protein-coding transcripts with and without antisense transcription.

A. Size distribution of lncRNAs, B. size distribution, C. number of exons, and D. position of introns in annotated transcripts with (black bars) and without (white bars) antisense transcription.

Figure 2. Genomic distribution of annotated and lncRNA transcripts.

Annotated genes from the BROAD database are depicted in blue and those that are expressed above a threshold of > = 50 reads in our combined datasets are depicted in black. The distribution of all lncRNAs is shown in green and antisense transcripts are shown in red. Large gaps in the gene annotation indicate centromeric regions.

Figure 3. Examples of sense/antisense transcript pairs annotated from RNA sequencing data and validated by qPCR.

Panels display the locations and distribution of RNAseq reads of sense protein-coding (black) and antisense lncRNA (pink) transcripts. RNAseq reads from the WT dark (D), light pulse (L) and temperature pulse (T) samples mapping to each locus are shown; read count scales differ. Below each panel, arrows represent sense (black) and antisense (pink) transcripts. Thick lines represent exons and thin lines introns. Grey boxes indicate the approximate region of each transcript amplified by qRTPCR. Reads are shown for the following sense transcripts and their complementary antisense RNAs: NCU04182 (coding for splicing factor 3 b subunit 4), NCU07268 (coding for a hypothetical protein with PAS domain), NCU02607 (coding for hypothetical protein), NCU07915 (coding for integral membrane protein), NCU09135 (coding for phosphatidylinositol phospholipase C) and NCU09136 (coding for a hypothetical protein. A single antisense transcript overlaps both NCU09135 and NCU09136. The two transcripts antisense to NCU09136 are separated by more than 200 nts.

Figure 4. qPCR verification of expression of selected sense/antisense pairs.

Expression of both the sense and antisense transcript for NCU04182, NCU07268, NCU02607, NCU07915, NCU09135 and NCU09136 in the WT is shown, after growth in the dark, and exposure to light and temperature pulses. Black bars indicate the protein-coding sense transcript and white bars indicate its antisense transcript. Error bars represent standard deviation. Statistical significance between light vs dark and temperature vs dark was determined using Student t test, * indicates p-value <0.05 and ** indicates p-value < = 0.005. n = 3.