Interplay of heavy chain introns influences efficient transcript splicing and affects product quality of recombinant biotherapeutic antibodies from CHO cells.

Introns are included in genes encoding therapeutic proteins for their well-documented function of boosting expression. However, mis-splicing of introns in recombinant immunoglobulin (IgG) heavy chain (HC) transcripts can produce amino acid sequence product variants. These variants can affect product quality; therefore, purification process optimization may be needed to remove them, or if they cannot be removed, then in-depth characterization must be carried out to understand their effects on biological activity. In this study, HC transgene engineering approaches were investigated and were successful in significantly reducing the previously identified IgG HC splice variants to <0.5%. Subsequently, a comprehensive evaluation was conducted to understand the influence of the different introns in the HC genes on the expression of recombinant biotherapeutic antibodies. The data revealed an unexpected cooperation between specific introns for efficient splicing, where intron retention led to significant reductions in IgG expression of up to 75% for some intron combinations. Furthermore, it was shown that HC introns could be fully removed without significantly affecting productivity. This work paves the way for future biotherapeutic antibody transgene design with regard to inclusion of HC introns. By removing unnecessary introns, transgene mRNA transcript will no longer be mis-spliced, thereby eliminating HC splice variants and improving antibody product quality.

Functions for fission yeast splicing factors SpSlu7 and SpPrp18 in alternative splice-site choice and stress-specific regulated splicing.

Budding yeast spliceosomal factors ScSlu7 and ScPrp18 interact and mediate intron 3'ss choice during second step pre-mRNA splicing. The fission yeast genome with abundant multi-intronic transcripts, degenerate splice signals and SR proteins is an apt unicellular fungal model to deduce roles for core spliceosomal factors in alternative splice-site choice, intron retention and to study the cellular implications of regulated splicing. From our custom microarray data we deduce a stringent reproducible subset of S. pombe alternative events. We examined the role of factors SpSlu7 or SpPrp18 for these splice events and investigated the relationship to growth phase and stress. Wild-type log and stationary phase cells showed ats1+ exon 3 skipped and intron 3 retained transcripts. Interestingly the non-consensus 5'ss in ats1+ intron 3 caused SpSlu7 and SpPrp18 dependent intron retention. We validated the use of an alternative 5'ss in dtd1+ intron 1 and of an upstream alternative 3'ss in DUF3074 intron 1. The dtd1+ intron 1 non-canonical 5'ss yielded an alternative mRNA whose levels increased in stationary phase. Utilization of dtd1+ intron 1 sub-optimal 5' ss required functional SpPrp18 and SpSlu7 while compromise in SpSlu7 function alone hampered the selection of the DUF3074 intron 1 non canonical 3'ss. We analysed the relative abundance of these splice isoforms during mild thermal, oxidative and heavy metal stress and found stress-specific splice patterns for ats1+ and DUF3074 intron 1 some of which were SpSlu7 and SpPrp18 dependent. By studying ats1+ splice isoforms during compromised transcription elongation rates in wild-type, spslu7-2 and spprp18-5 mutant cells we found dynamic and intron context-specific effects in splice-site choice. Our work thus shows the combinatorial effects of splice site strength, core splicing factor functions and transcription elongation kinetics to dictate alternative splice patterns which in turn serve as an additional recourse of gene regulation in fission yeast.

Ubiquitination-mediated interaction among domains is responsible for inhibition of RNA endonuclease activity of mRNA cycling sequence binding protein from L. donovani (LdCSBP).

In nearly complete absence of transcriptional regulation, messenger RNA (mRNA) turnover mediated through specific cis-elements plays a predominant role in the control of differential gene expression for the disease causing trypanosomatid parasites. In these organisms, the periodic accumulation of S-phase messages during cell cycle is determined by the presence of one or more copies of a conserved CAUAGAAG octanucleotide motif in the untranslated regions of mRNAs. In our previous studies, a multi-domain cycling sequence binding protein LdCSBP from Leishmania donovani was characterized, which binds specifically to the octamer-containing RNAs via its uniquely arranged CCCH-type Zn fingers and degrades them through its small MutS-related (Smr) endonuclease domain, indicative of its potential role in the turnover of the S-phase mRNAs. Interestingly, the protein is modified by the incorporation of a monoubiquitin residue, and the posttranslational modification inhibits its riboendonuclease activity. However, the mechanism of such inhibition was previously unknown. Here, we establish that the CCCH-type Zn finger domain is the site of ubiquitination in LdCSBP and the interaction of CUE domain of the protein with the ubiquitinated Zn finger domain is responsible for inhibition of its riboendonuclease activity. The findings elucidate an inhibitory mechanism of RNA cleavage through ubiquitination-mediated intramolecular interaction among domains of the enzyme. Furthermore, the riboendonuclease activity is inhibited by anti-leishmanial drug paromomycin suggesting that the regulation of RNA metabolism could be a target of the drug.