Figure 5

Matrin 3 stabilizes RRE-containing RNA. A) (top) Schematic representations of the differently sized mRNA transcripts produced during HIV-1 replication. The 9 kb (unspliced) and 4 kb (singly spliced) viral transcripts contain the RRE cis-element and require Rev protein for expression. (bottom) HeLa cells were transfected with HIV-1 molecular clone pNL4-3 and either pCMV-HA or HA-Matrin 3 plasmids. Northern blot analysis of whole cell RNA demonstrated increased expression of unspliced 9 kb HIV-1 transcript (lane 2). Relative changes in the expression of 9 kb and 1.8 kb HIV-1 RNAs in cells, with and without Matrin 3, are shown by the numbers on the right. B) Matrin 3 increased the stability and promoted the nuclear export of HIV-1 unspliced RNA. HeLa cells were transfected with pNL4-3 with (red) or without (green) Matrin 3. RNA was isolated from whole cell lysates as well as nuclear and cytoplasmic fractions. qRT-PCR analysis of HIV-1 RNA was performed using primers specific for spliced and unspliced viral transcripts [29]. Transfection of Matrin 3 (red) resulted in modestly increased amounts of HIV-1 unspliced transcripts in the cells (top left panel, total), and a much larger increase in the distribution of unspliced HIV-1 transcripts into cytoplasm (top right panel, cytoplasmic). As control, Matrin 3 did not affect the stability or the distribution of GAPDH mRNA (bottom panels, GAPDH).