A nuclear envelope protein linking nuclear pore basket assembly, SUMO protease regulation, and mRNA surveillance

The nuclear pore complex (NPC) is both the major conduit for nucleocytoplasmic trafficking and a platform for organizing macromolecules at the nuclear envelope. We report that yeast Esc1, a non-NPC nuclear envelope protein, is required both for proper assembly of the nuclear basket, a structure extending into the nucleus from the NPC, and for normal NPC localization of the Ulp1 SUMO protease. In esc1Δ cells, Ulp1 and nuclear basket components Nup60 and Mlp1 no longer distribute broadly around the nuclear periphery, but co-localize in a small number of dense-staining perinuclear foci. Loss of Esc1 (or Nup60) alters SUMO conjugate accumulation and enhances ulp1 mutant defects. Similar to previous findings with Mlp1, both Esc1 and Ulp1 help retain unspliced pre-mRNAs in the nucleus. Therefore, these proteins are essential for proper nuclear basket function, which includes mRNA surveillance and regulation of SUMO protein dynamics. The results raise the possibility that NPC-localized protein desumoylation may be a key regulatory event preventing inappropriate pre-mRNA export

Complete study demonstrating the absence of rhabdovirus in a distinct Sf9 cell line - Fig 3

<p>(A). RT-PCR of total RNA from Sf9 cells. Primers were designed to amplify 14 target sequences that cover 99.1% of the reported Sf-rhabdovirus RNA in an overlapping manner as indicated by the gene schematic. Sizes of 13 target sequences ranged from ~1.0 to 1.3 kb (lanes 1–13) and one target sequence was ~0.5 kb (lane 14). The sizes of amplicons matched the expected sizes of target sequences except for one amplicon (*, lane 7), which was amplified by primers targeting bp 6965–7134. (B). Sequencing of the amplicon revealed a deletion of 320 bp at position 6371–6690 in Sf-rhabdovirus RNA. The 320 bp deletion contains the 3’ region of ORF-X and a part of the intergenic region between ORF-X and ORF-L that includes rhabdovirus conserved transcription motifs for the X and L genes.</p

BLAST search of SfRV RNA sequence against GenBank EST database.

<p>EST clone sequences showing homology to SfRV RNA are aligned on the map and represented by colored circles; yellow circle, <i>Bombyx mori</i> (Bm5 or BmN cells, or Nnor); green circle, BmNPV infected BmN cells; blue circle, <i>Heliothis virescence</i>; black circle, <i>Aphid gossippi</i>; closed circle, sense strand sequence; open circle, anti-sense strand sequence.</p

BLAST analysis of SfRV RNA (GenBank, KF947078) against Sf21 cell genome shotgun sequence (GenBank, JQCY00000000.2).

<p>BLAST analysis of SfRV RNA (GenBank, KF947078) against Sf21 cell genome shotgun sequence (GenBank, JQCY00000000.2).</p

Identification of major protein components in the 1.14 g/ml sucrose density gradient fraction of Sf9 culture supernatant by LC MS/MS analysis.

<p>Eleven groups containing up to 3 stained protein bands were analyzed. The major protein identified per band is listed on the right.</p

qRT-PCR of sucrose density gradient fractions using SfRV L gene primers.

<p>Ct values were normalized to that of fraction 2 and are represented as a relative copy number of target SfRV L gene sequence. The photo shows the sucrose density gradient prior to fractionation. Left to right: top to bottom of the gradient.</p

Chimeric sequence of SfRV and Sf genome in four inverse PCR clones obtained from Sf9 DNA.

<p>Chimeric sequence of SfRV and Sf genome in four inverse PCR clones obtained from Sf9 DNA.</p

Electron microscopy of the 1.14 g/ml fraction.

<p>Bar indicates 100 μm in length. A, no incubation after loading the 1.14 fraction (diluted with distilled water) onto a grid. B, 1 min incubation after loading. Both grids were washed twice with distilled water and sample was stained with 1% uranyl acetate.</p