THOC2 likewise mislocalized to the cytoplasm of cells expressing Htt96Q or TDP-F4. Contrary to the β proteins, THOC2 now became secluded into separate aggregates, distinct from the Htt96Q or TDP-F4 inclusions. Partly, a fraction of THOC2 remained nuclear, especially in case of TDP-TDP-F4. The mislocalization and aggregation of THOC2 occurred in all cases of nuclear mRNA retardation in presence of cytoplasmic protein aggregates, as described above.
In cells expressing cytoplasmic β proteins, Htt96Q, and in part TDP-F4, mislocalized THOC2 was obviously unable to (re-)enter the nucleus. This may either occur due to sequestration of THOC2 into misfolded low- or high-molecular-weight structures, or possibly, due to defects in nuclear import.
Also nuclear mRNA processing and export proteins require a functional nuclear import on their own, at least once after their cytoplasmic synthesis, or continuously in case of shuttling factors.
Furthermore, cytoplasmic mislocalization of THOC2 was found in brains of R6/2 mice. Neurons that were recognized by antibodies against extended polyQ sequences (3B5H10) often contained cytoplasmic THOC2, which was missing at its natural nuclear localization.
Figure 84 | Brain sections of 9 week old wildtype and R6/2 mice were stained for THOC2 (green) with an antibody specific for extended polyQ sequences (3B5H10, red), and for nuclear DNA (blue). Neurons of R6/2 mice with visible aggregates often contain cytoplasmic THOC2, which is strongly enriched in the nuclei of unaffected cells. Representative images from 3 wildtype and 4 R6/2 mice.
siRNA-induced silencing of the transcriptional export complex protein THOC2 THOC2 is an essential protein of the transcriptional export complex (TREX). Inactivation of THOC2 e.g.
through misfolding, mislocalization, or recruitment into aggregates likely causes severe mRNA processing and transport problems (Chi 2013). Therefore, we were interested in the phenotype of cells with low levels of functional THOC2.
3 days after transfection of siRNA targeting human THOC2, cellular THOC2 levels were decreased to 13 ± 3% in HEK293T cells. At the same time, cytoplasmic mRNA levels vanished, while the nuclear mRNA speckles remained. Their size was comparable to snRNPs of wildtype cells (but significantly smaller compared to the protein aggregation-induced nuclear mRNA bodies; Figure 86).
However, silencing of THOC2 was sufficient to cause nuclear mRNA retention, resulting in a strong decrease of cytoplasmic mRNA levels. Control siRNA targeting firefly luciferase had no effect on cellular THOC2 levels or on mRNA distribution.
A
B
C
Figure 85 | Knockdown of THOC2 by siRNA leads to a nuclear export inhibition of mRNA. (A) HEK293T and (B) SH-SY5Y cells were transfected with siRNA targeting human THOC2 for 3 days. Cells were stained for mRNA (green), THOC2 (red), and nuclear DNA (blue). Knocking down THOC2 caused a strong reduction of cytoplasmic mRNA levels, but nuclear snRNPs remained wildtype-like. Differential interference contrast is shown in (B). Representative images of 3 independent experiments.
Scale bar length 10 µM. (C) Immunoblotting for THOC2 demonstrated the decrease in THOC2 levels after 3 days of siRNA treatment.
GAPDH levels shown for comparison.
Aggravated nuclear mRNA bodies in cells with pathogenic protein aggregation The nuclear mRNA bodies observed in cells with cytoplasmic protein aggregation were of strongly increased size and brightness in comparison to wildtype or THOC2 siRNA-treated cells. This result suggests that the presence of toxic protein aggregates in the cytoplasm caused a more severe phenotype with a potentially different composition of the nuclear mRNA structures.
Under selective knockdown of THOC2 by targeted siRNA, nuclear mRNA processing or cellular responses to missing mRNA export may still be functional. In the presence of toxic protein aggregation, additional defects such as insufficient processing of premature mRNAs or a disturbed nuclear RNA degradation may be an explanation for the increased size of the nuclear mRNA bodies.
Associated (misfolded?) proteins might furthermore increase the volume of the nuclear mRNA bodies, e.g. RNA binding and processing proteins.
Sole nuclear mRNA export inhibitions might have different consequences for mRNA fate. The mRNA might be rapidly degraded, e.g. by the exosome, a nuclear quality control RNase (Houseley 2006). Or the mRNA may accumulate for longer times in the nucleus, accidently or for temporal storage and subsequent export. Depending on the outcome, lowered or increased mRNA levels may be found.
To quantify the mRNA content of single cells, the complete mRNA fluorescence of was integrated (representing mainly nuclear mRNA in cells with cytoplasmic β protein aggregates) and compared to surrounding untransfected cells with a normal mRNA conent and distribution. Compared to wildtype cells, the integrated mRNA fluorescence of cells expressing cytoplasmic β proteins roughly doubled.
In addition, the mRNA content of cells expressing Htt96Q and TDP-F4 rose significantly (to ~160-175%; only cells with mRNA retention were analyzed for this quantification). Again, only a marginal increase in mRNA levels was found for cells expressing nuclear β proteins (~105-120%). The observed raise of cellular mRNA levels points towards a real, physical accumulation of mRNA in the nucleus. The mRNA was neither exported to the cytoplasm, nor immediately degraded in the nucleus. Protein misfolding and aggregation in the cytoplasm resulted in brightly fluorescent, nuclear bodies with substantially increased amounts of mRNA.
Figure 86 | mRNA distribution (green) in SH-SY5Y cells transfected with empty control plasmids, siRNA against THOC2, or β23-EGFP (red). The mRNA is distributed over the cytoplasm and concentrated in nuclear snRNPs of control cells. The cytoplasmic mRNA vanished in cells lacking THOC2 or expressing β23-EGFP. While the nuclear snRNPs in cells treated with siRNA against THOC2 appeared almost wildtype-like, size and brightness of the nuclear mRNA bodies were strongly increased in cells with protein aggregates.
It would be highly interesting to analyze the RNA and protein content of the nuclear mRNA bodies in cells with cytoplasmic protein aggregates. A profound analysis of mRNA sequences may reveal functional and defective steps during mRNA processing. Especially mRNA splicing might be affected, while the polyA tail appeared to be synthesized due to the usage of a fluorescent poly(d)T probe.
Identification of absent snRNP proteins or appereance of atypical factors should allow us to gain further insights into the mechanisms of nuclear mRNA body formation and associated cellular (dys-)functions.
Figure 87 | Single cell quantification of total mRNA. (A) SH-SY5Y cells transfected with NES-β23 and stained for mRNA (polyA RNA, green), NES-β23 (c-Myc antibodies, red), and nuclear DNA (DAPI, blue) 24 h after transfection. The cellular polyA RNA fluorescence of cells with NES-β23 aggregates was integrated (red dashed line) and set in ratio to the integrated polyA RNA fluorescence of untransfected cells (white dashed lines) on the same focal area. (B) Quantification of total cellular mRNA content of single cells with the indicated protein aggregates and a nuclear mRNA accumulation phenotype, in relation to untransfected cells set to 100% (≥3 independent experiments; n = 4-13 cells, each). Cells with β proteins directed to the cytoplasm, Htt96Q, or TDP-F4 show an increase in cellular mRNA content, originating from an mRNA accumulation in the nucleus. Cells with nuclear β protein aggregates show no significant increase of cellular mRNA levels.
A B
50%
75%
100%
125%
150%
175%
200%
225%
250%
re l. m RN A / c el l
*** *
* **
n.s. n.s .