Updated project metadata. RNA splicing and protein degradation systems allow the functional adaptation of the proteome in response to changing cellular contexts. However, the regulatory mechanisms connecting these processes remain poorly understood. Here, we show that impaired spliceosome assembly caused by USP39 deficiency leads to a pathogenic splicing profile characterized by the use of cryptic 5′ splice sites. To explore the interactions of USP39 and the effect of its depletion in HeLa cells, we performed complexome profiling of nuclear lysates. We observed most USP39 stably integrated into the tri-snRNP complex and there is almost no free nuclear protein. More importantly, the relative abundance of tri-snRNP spliceosome complex was impaired in USP39-depleted cells. As previous reports indicated, USP39 is a regulator of tri-snRNP stability and its depletion decreased the levels of assembled U4/U6.U5 complexes.